Hdv polyclonal antibody, preparation method thereof, anti-hdv total antibody detection method, kit and application

Abstract

The application relates to the field of biotechnology, in particular to an HDV polyclonal antibody and a preparation method thereof, an anti-HDV total antibody detection method, a kit and application. The application immunizes non-human animals with HDV recombinant antigens in which multiple HDV small antigens and three linear epitopes on the HDV small antigens are connected in series, and the obtained HDV polyclonal antibody has full epitope coverage, high sensitivity, and is used in cooperation with the HDV recombinant protein, so that the anti-HDV total antibody in a sample can be quickly, sensitively and accurately detected based on a competition method.

Description

Technical Field

[0001] This application relates to the field of biotechnology, and in particular to HDV polyclonal antibodies and their preparation methods, methods for detecting total anti-HDV antibodies, kits, and applications. Background Technology

[0002] Hepatitis D virus (HDV) is a defective satellite virus that cannot replicate independently and must rely on envelope proteins provided by hepatitis B virus (HBV) to assemble and release viral particles. The HDV genome is a single-stranded, negative-sense circular RNA, approximately 1676-1683 nucleotides long. The virus is a spherical particle with a diameter of approximately 36 nm.

[0003] Co-infection or co-infection with HDV and HBV (Hepatitis B Virus) leads to the most severe form of viral hepatitis, accelerating the progression of liver fibrosis, cirrhosis, and liver failure, and significantly increasing the risk of liver cancer. Therefore, timely and accurate diagnosis of HDV is crucial for patient prognosis management and public health.

[0004] Currently, laboratory diagnosis of HDV infection mainly relies on serological testing (such as detecting anti-HDV antibodies and HDV antigens) and molecular biological testing (such as detecting HDV RNA). Serological testing remains the primary method for clinical screening and epidemiological investigations due to its advantages of simplicity, low cost, and high throughput. High-performance antibodies are the core raw material for developing these serological test kits. However, there is a shortage of existing HDV antibody raw materials, and some antibodies obtained through immunization have low affinity and increased specific cross-reactivity due to immunogenic defects; for example, antibodies derived from synthetic peptides may not effectively recognize the spatial conformational epitopes of viral native proteins. These defects easily lead to false positive or false negative results in testing. Meanwhile, because the detection of HDV and hepatitis D has not received widespread attention, there is still a significant gap in the diagnosis of hepatitis D. There are few existing anti-HDV total antibody detection products, and most of the existing methods suffer from complexity and low detection efficiency. Summary of the Invention

[0005] Therefore, it is necessary to provide an HDV polyclonal antibody and its preparation method, a method for detecting total anti-HDV antibodies, a kit, and its application.

[0006] In a first aspect, a method for preparing HDV polyclonal antibodies is provided, the method comprising immunizing non-human animals with recombinant HDV protein as an immunogen and isolating the polyclonal antibodies produced by the non-human animals.

[0007] The HDV recombinant protein contains the following structure:

[0008] From N-terminus to C-terminus: [S-HDAg]-[P1]-[P2]-[P3];

[0009] S-HDAg is a small antigen of HDV;

[0010] P1 consists of amino acid residues 59 through 84 of the HDV small antigen.

[0011] P2 consists of amino acid residues from position 92 to position 114 of the HDV small antigen.

[0012] P3 consists of amino acid residues 149 to 172 of the HDV small antigen;

[0013] The position of the amino acid residue is determined with reference to the amino acid sequence shown in SEQ ID NO.1, following the direction from the N-terminus to the C-terminus, with the first amino acid residue at the N-terminus of the amino acid sequence shown in SEQ ID NO.1 as the first position.

[0014] In an optional embodiment, the amino acid sequence of the HDV small antigen is the amino acid sequence shown in SEQ ID NO.1, or an amino acid sequence that has at least 90% identity with the amino acid sequence shown in SEQ ID NO.1.

[0015] In an optional embodiment, the amino acid sequence of P1 is the amino acid sequence shown in SEQ ID NO.2, or an amino acid sequence that has at least 90% identity with the amino acid sequence shown in SEQ ID NO.2.

[0016] In an optional embodiment, the amino acid sequence of P2 is the amino acid sequence shown in SEQ ID NO.3, or an amino acid sequence that has at least 90% identity with the amino acid sequence shown in SEQ ID NO.3.

[0017] In an optional embodiment, the amino acid sequence of P3 is the amino acid sequence shown in SEQ ID NO.4, or an amino acid sequence that has at least 90% identity with the amino acid sequence shown in SEQ ID NO.4.

[0018] In an optional implementation, any two domains in the HDV recombinant protein are directly connected or connected via linkers.

[0019] In an optional embodiment, the HDV recombinant protein further contains a tag.

[0020] In an optional embodiment, the HDV recombinant protein is expressed by a prokaryotic system; more preferably, the HDV recombinant protein is expressed by an Escherichia coli expression system.

[0021] In an optional embodiment, the amino acid sequence of the HDV recombinant protein is shown in SEQ ID NO.5.

[0022] In an optional embodiment, the preparation method further includes collecting serum from non-human animals immunized with the recombinant HDV protein and then purifying it to obtain the HDV polyclonal antibody.

[0023] Secondly, an HDV polyclonal antibody prepared using the method for preparing HDV polyclonal antibodies described in the first aspect is provided.

[0024] Thirdly, the application of the HDV polyclonal antibody described in the second aspect is provided in any one of (i) to (v):

[0025] (i) Detection of total anti-HDV antibodies;

[0026] (ii) Prepare a kit for detecting total anti-HDV antibodies;

[0027] (iii) Detection, purification, or enrichment of HDV antigen;

[0028] (iv) Prepare kits for detecting, purifying or enriching HDV antigens;

[0029] (v) Prepare a diagnostic kit for HDV infection.

[0030] Fourthly, a competitive assay kit for detecting total anti-HDV antibodies is provided, comprising (i) the recombinant HDV protein as defined in the first aspect; and (ii) the HDV polyclonal antibody as described in the second aspect.

[0031] In an optional embodiment, the HDV recombinant protein is linked to a solid-phase support, and the HDV polyclonal antibody is labeled with a signaling agent.

[0032] In an optional embodiment, the HDV polyclonal antibody is labeled with a chemiluminescent reagent.

[0033] In an optional embodiment, the competitive method-based anti-HDV total antibody detection kit is used for chemiluminescent immunoassay and includes magnetic microparticles coated with the HDV recombinant protein, the HDV polyclonal antibody labeled with acridine ester, magnetic microparticle diluent, and acridine diluent.

[0034] Fifthly, a competitive method for detecting total anti-HDV antibodies is provided. This method includes using the HDV recombinant protein defined in the first aspect to bind with the total anti-HDV antibodies in the sample, then using the HDV polyclonal antibody described in the second aspect to bind with the vacant epitopes of the HDV recombinant protein in the reaction system, and then obtaining the signal intensity of the signal linked to the HDV polyclonal antibody, wherein the signal intensity is negatively correlated with the content of total anti-HDV antibodies.

[0035] In an optional embodiment, the HDV recombinant protein is linked to a solid-phase support, and the HDV polyclonal antibody is labeled with a signaling agent.

[0036] In an optional embodiment, the HDV polyclonal antibody is labeled with a chemiluminescent reagent.

[0037] In an optional embodiment, the HDV recombinant protein is linked to magnetic microparticles, and the HDV polyclonal antibody is labeled with acridine ester.

[0038] This application utilizes a recombinant HDV protein with multiple antigenic epitopes as an antigen, enabling the preparation of a highly active HDV polyclonal antibody with comprehensive epitope recognition through immunization. This HDV polyclonal antibody exhibits comprehensive epitope coverage and high sensitivity, making it suitable for the detection of total anti-HDV antibodies and the preparation of detection kits, demonstrating significant clinical value and reliability. The competitive anti-HDV total antibody detection kit developed in this application utilizes the aforementioned recombinant HDV protein and HDV polyclonal antibody to rapidly, sensitively, and accurately detect total anti-HDV antibodies in samples. It avoids the risk of false negatives / weak positives in samples with extremely high anti-HDV antibody levels due to the "hook effect" and limited coverage of recombinant antigenic epitopes in the double-antigen sandwich method and indirect method, especially for rapid and sensitive detection of total HDV antibodies in clinical samples, with no false positives or missed detections. The anti-HDV polyclonal antibody developed in this application promotes the product development, upgrading, and application of hepatitis D detection; it can be widely used in HDV patient screening and ultimately support the prevention and treatment of hepatitis D. Attached Figure Description

[0039] To more clearly illustrate the technical solutions in the embodiments and examples of this application, and to more completely understand this application and its beneficial effects, the accompanying drawings used in the description of the embodiments or examples will be briefly introduced below. Obviously, the drawings described below are merely some embodiments of this application. Those skilled in the art can obtain other drawings based on these drawings without any creative effort.

[0040] Figure 1 This is an SDS-PAGE image of the HDV polyclonal antibody prepared using New Zealand male rabbits in Example 2;

[0041] Figure 2The standard curve was established for the chemiluminescent immunoassay of anti-HDV total antibody constructed in Example 4. Detailed Implementation

[0042] The present application will be further described in detail below with reference to the accompanying drawings, embodiments, and examples. It should be understood that these embodiments and examples are for illustrative purposes only and are not intended to limit the scope of the present application. The purpose of providing these embodiments and examples is to enable a more thorough and comprehensive understanding of the disclosure of the present application. It should also be understood that the present application can be implemented in many different forms and is not limited to the embodiments and examples described herein. Those skilled in the art can make various modifications or alterations without departing from the spirit of the present application, and the equivalent forms obtained also fall within the protection scope of the present application. For example, features described or illustrated as part of one embodiment can be combined in a suitable manner in another embodiment to produce new embodiments. Furthermore, numerous details are set forth in the following description to provide a fuller understanding of the present application. It should be understood that the present application can be implemented without one or more of these details.

[0043] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein in the specification of this application is for descriptive purposes only and is not intended to be limiting of the application.

[0044] Unless otherwise stated or in case of contradiction, the terms or phrases used herein shall have the following meanings:

[0045] The terms “and / or,” “or / and,” and “and / or” as used herein include any one of two or more of the related listed items, as well as any and all combinations of the related listed items. “Any and all combinations” includes any two related listed items, any more related listed items, or a combination of all related listed items. For example, “A and / or B” includes three parallel options: A, B, and “a combination of A and B.”

[0046] In this application, the terms "multiple", "various", "multiple times", "several", "several", etc., unless otherwise specified, refer to a quantity greater than or equal to 2. For example, "one or more" means one or more or more.

[0047] In this application, "optionally", "optional", and "optional" mean that something is optional, that is, it means that it is selected from either "with" or "without".

[0048] In this application, the technical features or solutions described in open-ended language include both closed-ended technical features or solutions consisting of the listed contents and open-ended technical features or solutions that include the listed contents.

[0049] In this application, where the method flow involves multiple steps, unless otherwise explicitly stated herein, there is no strict order restriction on the execution of these steps; they can be executed in any order other than those described. Moreover, any step may include multiple sub-steps or multiple stages, which are not necessarily completed at the same time, but can be executed at different times, and their execution order is not necessarily sequential, but can be performed alternately or simultaneously with other steps or parts of the sub-steps or stages of other steps.

[0050] In this application, the terms "first aspect," "second aspect," "third aspect," "fourth aspect," etc., are used for descriptive purposes only and should not be construed as indicating or implying relative importance or quantity, nor should they be construed as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first," "second," "third," "fourth," etc., serve only as a non-exhaustive enumeration and should be understood not to constitute a closed limitation on quantity.

[0051] In this application, "HDV small antigen" refers to the small hepatitis D virus antigen (S-HDAg), which is a core protein in the hepatitis D virus (HDV) replication cycle and consists of 195 amino acids encoded by the unique open reading frame of the viral genome.

[0052] In this application, unless otherwise specified, the amino acid positions of the HDV small antigen refer to the amino acid sequence shown in SEQ ID NO.1, with the positions arranged from the N-terminus to the C-terminus, and the first amino acid residue at the N-terminus of the amino acid sequence shown in SEQ ID NO.1 designated as position 1. However, it should be noted, and those skilled in the art will understand, that different sequences can have different numbering systems, for example, if additional amino acid residues are added or removed compared to SEQ ID NO.1. Therefore, when referring to a specific amino acid residue by its number, this description is not limited to the amino acid precisely located at that numbered position when counting from the amino acid sequence of SEQ ID NO.1, but also refers to equivalent / corresponding amino acid residues in any and all sequences, the equivalent / corresponding positions of which can be obtained by comparison with the following definition of "identity," even if the residue is not at the same precise numbered position. For example, if the sequence is shorter or longer than SEQ ID NO.1, or has insertions or deletions compared to SEQ ID NO.1. For example, if sequence X is missing two amino acid residues at the N-terminus compared to the sequence in SEQ ID NO. 1, then position 1 of sequence X is equivalent to position 3 of the sequence in SEQ ID NO. 1. Unless otherwise specified, the reference to the amino acid position in sequence X refers to position 3 when the amino acid position in sequence X is used as a reference. It should be noted that when HDV small antigen or a portion thereof is fused with other polypeptides, the amino acid sequence of the HDV small antigen in the fusion protein is used as a reference sequence. For example, if the fusion protein contains HDV small antigen starting at position 101, then position 3 of the HDV small antigen portion in the fusion protein (i.e., position 103 of the fusion protein) is equivalent to position 3 of the sequence in SEQ ID NO. 1.

[0053] In this application, a "domain" refers to a specific region in a molecule that folds into a relatively independent structural unit in three-dimensional space, possessing specific functions and stability. A domain typically consists of one or more continuous portions of a peptide chain. These portions may not be continuous in the amino acid sequence of the protein, but they are close to each other in the three-dimensional structure, folding into a relatively independent structural unit. Generally, a domain is responsible for a single functional property and, in many cases, can be added to, removed from, or transferred to other molecules without losing the function of the rest of the molecule and / or the domain itself. The domains of the HDV recombinant protein in this application include at least S-HDAg, P1, P2, and P3, and optionally a tag.

[0054] In this application, peptide, polypeptide, and protein are not strictly distinguished and may be used interchangeably in some cases. Generally, peptides refer to polymers composed of amino acids linked by peptide bonds, whether naturally occurring or synthetic. All polypeptide sequences are written according to generally accepted conventions, with the α-N-terminal amino acid residue on the left and the α-C-terminal amino acid residue on the right. When used herein, the term "N-terminus" refers to the free α-amino group of an amino acid in a polypeptide, and the term "C-terminus" refers to the free α-carboxylic acid terminus of an amino acid in a polypeptide. A polypeptide ending with a group at the N-terminus refers to a polypeptide carrying a group on the α-amino nitrogen of an N-terminal amino acid residue. An amino acid ending with a group at the N-terminus refers to an amino acid carrying a group on its α-amino nitrogen.

[0055] In this application, the term "identity" percentage refers to the degree to which the amino acids of two polypeptides are identical at equivalent positions when two sequences are optimally aligned. The alignment of amino acid sequence identity percentages can be performed using various methods within the art, such as software well-known in the art, including BLAST, BLAST-2, ALIGN, MEGALIGN (DNASTAR), CLUSTALW, or CLUSTAL OMEGA. Those skilled in the art can determine appropriate parameters for the aligned sequences, including any algorithms required to achieve maximum alignment of the full length of the compared sequences.

[0056] In optional embodiments, the sequence that is identical to the target sequence herein differs from the target sequence from conserved amino acid substitutions. The term "conserved amino acid substitution" refers to the replacement of one amino acid residue with another amino acid residue that is physicochemically similar, such that the substitution does not alter or minimally alters the properties and function of the entire polypeptide or protein. Conserved amino acid substitution is well known to those skilled in the art. Families of amino acid residues with similar side chains are known in the art, including amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Special forms of conserved amino acid substitutions include those that replace amino acids that are not among the normal 20 amino acids encoded by the genetic code.

[0057] In this application, the term "epitope" refers to any antigenic determinant on an antigen that is bound to the complementary site of an antibody. An antigenic determinant is typically a specific chemical group with a defined composition and structure. Epitopes can be linear (i.e., continuous) or conformational (i.e., consisting of spaced-apart amino acid residues, discontinuous). An epitope defines the minimum binding site of an antibody and is therefore a specific target for the antibody or its antigen-binding fragment. Epitopes can be determined by any method well known in the art, such as conventional immunoassays, antibody competitive binding assays, or X-ray crystallography or related structural assays (e.g., nuclear magnetic resonance spectroscopy).

[0058] In this application, the term "linker" refers to a part used to connect any two molecules. A linker can be a small molecule compound, a polynucleotide, or a polypeptide, or a combination thereof. Alternatively, the linker is a linking peptide, which can be any linking peptide known in the art, such as a rigid linking peptide or a flexible linking peptide. An exemplary linking peptide is (G... x S) n x is an integer from 1 to 5, and n is an integer from 1 to 6.

[0059] In this application, the term "signaling substance" refers to a substance capable of providing a detectable signal that can be directly observed with the naked eye or detected by conventional instruments acceptable in the art. The signaling substance can directly provide a signal, such as color (e.g., colloidal gold, colored microspheres), fluorescence (fluorescent molecules), magnetism, radiation, or luminescence; or it can indirectly provide a signal through a subsequent reaction involving the signaling substance, such as a signal generated by an electrochemiluminescence reaction or an enzyme-catalyzed chemiluminescence reaction. Examples of signaling substances include, but are not limited to, one or more of chemiluminescent reagents, fluorescent labels, quantum dots, radionuclides, and paramagnetic ions. Chemiluminescent reagents can emit light directly or through an enzyme-catalyzed luminescence reaction or an electrochemical reaction. In optional embodiments, the chemiluminescent reagent includes, but is not limited to, at least one of acridine esters and their derivatives, luminol and its derivatives, luciferin, ruthenium bipyridine and its derivatives, dioxane and its derivatives, rofenine and its derivatives, and peroxate and its derivatives. In optional embodiments, the chemiluminescent reagent further includes a catalytic enzyme for the enzymatic luminescence reaction, including but not limited to horseradish peroxidase, alkaline phosphatase, β-galactosidase, and glucose oxidase.

[0060] In this application, the term "solid-phase support" refers to a solid support used in immunoassay techniques to bind and immobilize antibodies or antigens. It immobilizes immunoreactants on its surface through physical adsorption or chemical coupling, and is not easily detached after repeated washing and long-term storage, without affecting the immunoreactivity of the immobilized molecules. Exemplary solid-phase supports include, but are not limited to, microparticles, microtubes, columns, nitrocellulose membranes, chromatography matrices, side-flow devices, or microfluidic devices; more specifically, they may be, but are not limited to, magnetic beads, enzyme-labeled wells, immunochromatographic test strips, or other materials. The chromatography matrix may be any chromatography matrix known in the art, including, but not limited to, polystyrene, polysaccharide polymers, or silica gel.

[0061] In this application, the term "non-human animal" refers to all animals other than humans that belong to the animal kingdom in biological taxonomy. Examples of non-human animals include, but are not limited to, mice, rats, rabbits, goats, sheep, horses, chickens, guinea pigs, hamsters, cattle, pigs, dogs, cats, rhesus monkeys, cynomolgus monkeys, camels, alpacas, sharks, ducks, geese, and donkeys.

[0062] In one aspect, some embodiments provide a method for preparing an HDV polyclonal antibody, the method comprising immunizing a non-human animal with a recombinant HDV protein as an immunogen and isolating the polyclonal antibody produced by the non-human animal.

[0063] HDV recombinant protein contains the following structure:

[0064] From N-terminus to C-terminus: [S-HDAg]-[P1]-[P2]-[P3];

[0065] S-HDAg is a small antigen of HDV;

[0066] P1 consists of amino acid residues 59 through 84 of the HDV small antigen.

[0067] P2 consists of amino acid residues from position 92 to position 114 of the HDV small antigen.

[0068] P3 consists of amino acid residues 149 to 172 of the HDV small antigen;

[0069] The position of the amino acid residues is determined with reference to the amino acid sequence shown in SEQ ID NO.1, following the direction from the N-terminus to the C-terminus, with the first amino acid residue at the N-terminus of the amino acid sequence shown in SEQ ID NO.1 as the first position.

[0070] In an optional embodiment, the amino acid sequence of the HDV small antigen is the amino acid sequence shown in SEQ ID NO.1, or an amino acid sequence that has at least 90% (e.g., but not limited to at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity with the amino acid sequence shown in SEQ ID NO.1.

[0071] In an optional embodiment, the amino acid sequence of P1 is the amino acid sequence shown in SEQ ID NO.2, or an amino acid sequence that has at least 90% (e.g., but not limited to at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity with the amino acid sequence shown in SEQ ID NO.2.

[0072] In an optional embodiment, the amino acid sequence of P2 is the amino acid sequence shown in SEQ ID NO.3, or an amino acid sequence that has at least 90% (e.g., but not limited to at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity with the amino acid sequence shown in SEQ ID NO.3.

[0073] In an optional embodiment, the amino acid sequence of P3 is the amino acid sequence shown in SEQ ID NO.4, or an amino acid sequence that has at least 90% (e.g., but not limited to at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity with the amino acid sequence shown in SEQ ID NO.4.

[0074] In an optional implementation, any two domains in the HDV recombinant protein are directly linked or linked via linkers. Any two linkers in the HDV recombinant protein may be the same or different.

[0075] In an optional implementation, the HDV recombinant protein also contains a tag.

[0076] In an optional embodiment, the HDV recombinant protein is expressed using a prokaryotic system; in a further optional embodiment, the HDV recombinant protein is expressed using an E. coli expression system.

[0077] In an optional embodiment, the amino acid sequence of the HDV recombinant protein is shown in SEQ ID NO. 5. In a further optional embodiment, the amino acid sequence of the HDV recombinant protein is shown in SEQ ID NO. 5, and it is a recombinant protein expressed by an E. coli expression system.

[0078] In an optional embodiment, the preparation method further includes collecting serum from non-human animals immunized with recombinant HDV protein and then purifying it to obtain HDV polyclonal antibodies.

[0079] Secondly, the method for preparing HDV polyclonal antibodies as described in the first aspect is also provided.

[0080] In an optional implementation, the HDV polyclonal antibody is a polyclonal antibody derived from sheep serum or rabbit serum.

[0081] Thirdly, the application of HDV polyclonal antibodies in any of (i) to (v) is also provided:

[0082] (i) Detection of total anti-HDV antibodies: The HDV polyclonal antibody in the second aspect can be used as a competing antibody with the total anti-HDV antibody in the sample in the reaction system, and the total anti-HDV antibody is detected based on the principle of competition.

[0083] In an optional implementation, detecting total anti-HDV antibodies is not for diagnostic or therapeutic purposes.

[0084] (ii) Preparation of a kit for detecting total anti-HDV antibodies: Based on the principle of (i), the HDV polyclonal antibodies of the second aspect can be used to construct a kit for detecting total anti-HDV antibodies.

[0085] (iii) Detection, purification or enrichment of HDV antigens: HDV polyclonal antibodies can be used as capture antibodies or detection antibodies to establish immunological methods for detecting HDV antigens in samples through immunoassay methods, such as, but not limited to, enzyme-linked immunosorbent assay (ELISA), chemiluminescent immunoassay (CLIA) or immunochromatography. They can also be combined with solid-phase carriers to form immunoadsorbents for enrichment and purification of HDV antigens.

[0086] In an optional implementation, the detection of HDV antigen is not for diagnostic or therapeutic purposes.

[0087] (iv) Prepare kits for the detection, purification or enrichment of HDV antigens, based on the application in (iii), which can also be used to prepare kits for the detection, purification or enrichment of HDV antigens.

[0088] (v) Preparation of HDV infection diagnostic kits. HDV polyclonal antibodies can be used as competitive antibodies, signal-labeled secondary antibodies, capture antibodies, quality control products, and positive controls in the detection system to participate in the construction of immunoassay kits for the diagnosis of HDV infection. HDV polyclonal antibodies can be used in combination with the aforementioned HDV recombinant protein. Polyclonal antibodies obtained by immunization with HDV recombinant protein can also be used as standards or reference products for performance evaluation and quality control of the kits.

[0089] Fourthly, a competitive assay kit for detecting total anti-HDV antibodies is provided, comprising (i) the HDV recombinant protein as defined in the first aspect, which serves as an antigen for capturing total anti-HDV antibodies in a sample to be tested; and (ii) the HDV polyclonal antibody as defined in the second aspect, which serves as an antibody that competes with the total anti-HDV antibodies in the sample for binding to the antigenic epitopes on the HDV recombinant protein.

[0090] In an optional implementation, the HDV recombinant protein in the kit is linked to a solid-phase support, preferably magnetic microparticles.

[0091] In an optional implementation, the HDV polyclonal antibody in the kit is labeled with a signaling agent.

[0092] In an optional implementation, the HDV polyclonal antibody in the kit is labeled with a chemiluminescent reagent.

[0093] In optional embodiments, the kit can be used in generally acceptable immunoassay methods, including but not limited to chemiluminescent immunoassay (CLIA), immunofluorescence staining, flow cytometry, immunoblotting, immunohistochemistry, ELISA, immunochromatography, or immunomagnetic beads. Those skilled in the art can formulate reagents or other reagents in the kit according to the corresponding detection method, and this application does not limit this. Examples of reagents include, but are not limited to, one or more of the following: buffer reagents, salts, secondary antibodies, chromogenic substrates, blocking solutions, washing solutions, solvents, elution solutions, coupling agents, negative controls, positive controls, standards, quality controls, and labels.

[0094] In an optional embodiment, the kit is used for chemiluminescent immunoassay and includes magnetic microparticles coated with recombinant HDV protein, HDV polyclonal antibody labeled with acridine ester, magnetic microparticle diluent, and acridine diluent.

[0095] Fifthly, a competitive assay method for detecting total anti-HDV antibodies is provided. This method, based on the competitive assay principle, employs a two-step detection procedure. First, the HDV recombinant protein from the first step binds to the total anti-HDV antibody in the sample. Then, the HDV polyclonal antibody from the second step binds to the vacant epitopes of the HDV recombinant protein in the reaction system. Two complexes are formed in the system: "HDV recombinant protein-anti-HDV total antibody" and "HDV recombinant protein-HDV polyclonal antibody". The signal intensity of the signal molecule linked to the HDV polyclonal antibody is then measured. The signal intensity is negatively correlated with the content of the total anti-HDV antibody; that is, the higher the signal intensity, the higher the content of the "HDV recombinant protein-HDV polyclonal antibody" complex, and the lower the content of the "HDV recombinant protein-anti-HDV total antibody" complex. The detection results are then qualitatively, semi-quantitatively, or quantitatively determined based on the signal intensity.

[0096] In an optional implementation, the HDV recombinant protein is linked to a solid-phase carrier.

[0097] In an optional implementation, the HDV polyclonal antibody is labeled with a signaling agent.

[0098] In an optional implementation, the HDV polyclonal antibody is labeled with a chemiluminescent reagent.

[0099] In an optional embodiment, the HDV recombinant protein is linked to magnetic microparticles, and the HDV polyclonal antibody is labeled with acridine ester. A complex of "magnetically microparticle-coated HDV recombinant protein - anti-HDV total antibody" and a complex of "magnetically microparticle-coated HDV recombinant protein - acridine ester-labeled HDV polyclonal antibody" are formed in the reaction system. After the reaction, only the complex of "magnetically microparticle-coated HDV recombinant protein - acridine ester-labeled HDV polyclonal antibody" emits light. Therefore, the content of the complex of "magnetically microparticle-coated HDV recombinant protein - anti-HDV total antibody" is negatively correlated with the luminescence intensity.

[0100] In an optional implementation, the anti-HDV total antibody detection method is for non-diagnostic and non-therapeutic purposes.

[0101] The following are some examples.

[0102] The embodiments of this application will be described in detail below with reference to some examples. It should be understood that these embodiments are only for illustrating this application and are not intended to limit the scope of this application. For experimental methods in the following embodiments where conditions are not specified, please refer to the guidelines given in this application first, or follow experimental manuals or conventional conditions in the art, or follow the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0103] In the following examples, the measurement parameters of the raw material components may have slight deviations within the weighing accuracy range unless otherwise specified. Temperature and time parameters are subject to acceptable deviations due to instrument testing accuracy or operational precision.

[0104] Example 1

[0105] Prokaryotic expression and purification of recombinant antigen HDV-SE:

[0106] 1. HDV recombinant antigen HDV-SE design:

[0107] The HDV recombinant antigen HDV-SE has the following structure from the N-terminus to the C-terminus: [His tag]-[S-HDAg]-[P1]–[P2]–[P3];

[0108] S-HDAg is a small antigen of HDV, and its amino acid sequence is shown in SEQ ID NO.1.

[0109] SEQ ID NO.1:

[0110] MSRPEGRKNRGGREEVLEQWVSGRKKLEELERDLRKVKKKIKKLEDEHPWLGNIKGILGKKDKDGEGAPPAKRARTDQMEVDSGPRKRPSRGGFTDKERQDHRRRKALENKRKQLSAGGKNLSKEEEEELRRLTEEDERRERRIAGPQVGGVNPLEGGTRGAPGGGFVPSMQGVPESPFTRTGEGLDIRGSQGFP.

[0111] His tag is a His tag sequence that is inserted downstream of the N-terminal M (methionine residue) of the HDV small antigen to enable His tag expression. The His tag sequence can be used for subsequent protein purification.

[0112] P1, P2, and P3 are three linear epitopes in the HDV small antigen. P1 is aa59~84, and its amino acid sequence is shown in SEQ ID NO.2; P2 is aa92~114, and its amino acid sequence is shown in SEQ ID NO.3; P3 is aa149~172, and its amino acid sequence is shown in SEQ ID NO.4.

[0113] SEQ ID NO.2: GKKDKDGEGAPPAKRARTDQMEVDSG;

[0114] SEQ ID NO.3: GGFTDKERQDHRRRKALENKRKQ;

[0115] SEQ ID NO. 4: VGGVNPLEGGTRGAPGGGFVPSMQ.

[0116] S-HDAg and P1, P1 and P2, and P2 and P3 are all connected by connectors GGGGSGGGGSGGGGS (SEQ ID NO. 6).

[0117] The amino acid sequence of the HDV recombinant antigen HDV-SE is shown in SEQ ID NO.5.

[0118] SEQ ID NO.5:

[0119] MHHHHHHHHHHSRPEGRKNRGGREEVLEQWVSGRKKLEELERDLRKVKKKIKKLEDEHPWLGNIKGILGKKDKDGEGAPPAKRARTDQMEVDSGPRKRPSRGGFTDKERQDHRRRKALENKRKQLSAGGKNLSKEEEEELRRLTEEDERRERRIAGPQVGGV NPLEGGTRGAPGGGFVPSMQGVPESPFTRTGEGLDIRGSQGFPGGGGSGGGGSGGGGSGKKDKDGEGAPPAKRARTDQMEVDSGGGGGSGGGGSGGGGSGGFTDKERQDHRRRKALENKRKQGGGGSGGGGSGGGGSVGGVNPLEGGTRGAPGGGFVPSMQ.

[0120] 2. Plasmid construction: pET28a-S-HDAg-P1-P2-P3 expression clone was constructed, and its amino-terminal sequence is shown in SEQ ID NO.5.

[0121] 3. Plasmid Transformation: Transform the obtained vector into *E. coli* BL21, including the following steps: Thaw competent cells in an ice-water bath. Once the cells are freshly thawed, add the plasmid to the cells and mix gently. Incubate in an ice-water bath for 30 minutes. Heat shock at 42°C for 60 seconds. Incubate in an ice-water bath for 2 minutes without shaking. Add 500 μL of sterile LB medium. Incubate at 37°C with shaking at 150-200 rpm for 60 minutes. Spread 50-100 μL of the bacterial culture onto an LB agar plate containing an appropriate concentration of antibiotic resistance to the transformed plasmid. After the liquid has evaporated, invert the plate and incubate at 37°C for 12-16 hours.

[0122] 4. Protein Expression: Single colonies were picked and inoculated into LB medium containing 50 μg / mL kanamycin. The bacteria were cultured at 37°C with shaking at 200 rpm until the logarithmic growth phase (OD200). 600 =0.4~0.8). Remove the shake flask, add 1M IPTG in a clean bench to bring the final IPTG concentration to 0.5 mM; set the shaker temperature to 16℃ and the rotation speed to 200 rpm, and continue culturing for 24 h. Shaking culture at 16℃ induces BL21 protein expression, which greatly maintains the stability of the protein structure, increases the proportion of soluble recombinant antigen, and reduces the proportion of inclusion bodies.

[0123] 5. Protein purification: Centrifuge at 9000 rpm at 4℃ for 5 min, discard the supernatant, add lysis buffer (Triton X-405 added to the lysis buffer) and sonicate to lyse the bacterial cells. Centrifuge at 12000 rpm at 4℃ for 30 min, discard the precipitate, and collect the supernatant. Attach Ni to the supernatant. 2+ Protein purification is performed using an NTA column. After initial affinity purification, the obtained antibody is concentrated to a suitable concentration (1–10 mg / mL) and centrifuged or filtered to remove particulate matter. Sephadex G-25 or other molecular sieves are selected; the column volume is typically 30%–50% of the sample volume; the loading volume should not exceed 5% (fine separation) or 30% (desalting) of the column bed volume; isocratic elution is used, and the main peak is collected, avoiding the collection of tailing or prematurely eluted peaks. The collected antibody is ultrafiltered, centrifuged, and its concentration determined, then aliquoted and stored at -20°C.

[0124] Example 2

[0125] HDV polyclonal antibodies were prepared using male New Zealand rabbits.

[0126] 1. Immunization of animals: Two male New Zealand rabbits were acclimatized in a sterile animal room for 2 weeks. Approximately 2 mg of HDV-SE protein prepared in Example 1 was added to 2 mL of PBS, and 2 mL of Freund's complete adjuvant was added to emulsify the mixture as an antigen. The mixture was then subcutaneously injected into the male New Zealand rabbits at multiple sites, with an injection dose of 1 mL per rabbit. The initial immunization used Freund's complete adjuvant. Two weeks later, a booster immunization was performed by emulsifying half the amount of recombinant protein used in the initial immunization with an equal amount of Freund's incomplete adjuvant and subcutaneously injecting the mixture into the male New Zealand rabbits. A total of 3 booster immunizations were performed, with each booster immunization occurring at an interval of 2 weeks.

[0127] 2. Serum collection: Seven days after the last immunization, blood was collected from the carotid artery of male New Zealand rabbits. The blood was left to stand at room temperature for 2 hours, then incubated overnight at 4°C. After centrifugation at 4°C and 4000 rpm for 30 minutes, the supernatant was collected and treated at 56°C for 30 minutes to inactivate complement. The supernatant was purified using a Protein A column to obtain polyclonal antibodies against HDV, which were then aliquoted and stored at 80°C.

[0128] 3. Antibody titer detection: Antibody titer is determined by indirect ELISA detection method.

[0129] 4. Antibody specificity identification: Antibody specificity was determined by Western blot analysis, and the results are as follows: Figure 1 As shown.

[0130] Example 3

[0131] Preparation of HDV polyclonal antibodies using Boer goats:

[0132] 1. Immunization of animals: Boer goats were immunized with 10 mg of HDV-SE antigen prepared in Example 1 as the immunogen, with Freund's complete adjuvant added, by subcutaneous and intradermal multiple-point injection. Every 2 weeks, booster immunization was performed with Freund's incomplete adjuvant, by subcutaneous and intradermal multiple-point injection. After the three immunizations, blood was collected for antibody titer determination. Immunization was terminated when the antibody titer reached the required level.

[0133] 2. Serum Collection: After immunization is terminated, blood from Boer goats is collected. After natural coagulation, the supernatant is obtained by centrifugation. 20%–50% ammonium sulfate is added to the antiserum for precipitation. The precipitate is collected by centrifugation and redissolved in 20 mM–100 mM PBS (pH 7.2–8.0). The affinity packing material is washed with 10 mM–1 M citrate buffer (pH 2.5–4.0) until an elution peak appears. The elution peak is collected, and 1 M Tris-HCl (pH 8.0) is added as a neutralization buffer until the pH is between 6.5 and 7.5. The collected eluent is then dialyzed against 100 mM PBS buffer (pH 7.4) to prepare the specific HDV goat polyclonal antibody. The antibody is aliquoted and stored at -80°C.

[0134] 3. Antibody titer and specificity detection:

[0135] The titer and specificity of specific HDV sheep polyclonal antibodies were determined using enzyme-linked immunosorbent assay (ELISA). The steps are as follows:

[0136] (1) Coating: Dilute 1 mg / mL of specific HDV goat polyclonal antibody with carbonate buffer 1000 times and make a blank control group. Add 100 μL / well to the microplate and incubate overnight in a water bath at 37°C.

[0137] (2) Washing: Pour out the liquid in the well, set the plate washer parameters to add 300 μL of deionized water per well, wash the plate twice, and then spin dry the washing liquid.

[0138] (3) Sealing: Add 120 μL of sealing liquid to each well, seal at 37°C for 3 hours, spin dry the liquid in the well, and place in an oven at 37°C for 1 hour until dry.

[0139] (4) The enzyme-labeled HDV-SE recombinant antigen and real HDV positive antigen compete easily: The enzyme-labeled antigen is serially diluted; the HDV positive material is diluted with PBS to prepare a high-value antigen competition solution, and stored at 4℃ for later use. Titer column: Add 100 μL of blank dilution and 100 μL of serially diluted enzyme-labeled antigen to each well, and add PBS to the last two wells as a blank control; Inhibition column: Add 20 μL of high-value antigen competition solution and 100 μL of serially diluted enzyme-labeled HDV-SE recombinant antigen to each well, and add PBS dilution to the last two wells as a blank control; After shaking, incubate at room temperature for 40 min, and wash the plate 6 times.

[0140] (5) Color development: Add 100 μL of TMB color development solution to each well and develop the color at room temperature for 20 min. Then add 100 μL of stop solution (10% H2SO4) to each well.

[0141] (6) Reading determination: The absorbance (OD) was read using an ELISA reader at a wavelength of 450 nm. The specific HDV goat polyclonal antibody with an absorbance value in the range of 1.0 to 1.5 is the titer of the antibody. Under the same positive competitive solution concentration, the higher the inhibition rate, the higher the sensitivity of the antibody to HDV.

[0142] Example 4

[0143] Establishment of a chemiluminescent immunoassay for total anti-HDV antibodies:

[0144] 1. Preparation of HDV antigen-coated magnetic nanobeads: Take 50 mg of carboxylated magnetic beads (particle size 0.05~1 μm) suspension, magnetically separate to remove the supernatant, resuspend in 0.02 M, pH 5.5 MES buffer, add 0.5~2.0 mL of freshly prepared 10 mg / mL EDC aqueous solution to activate the carboxyl groups on the surface of the magnetic beads, and add 3~5 mg of HDV-SE antigen. Suspend at room temperature for 2~10 h, magnetically separate to remove the supernatant, resuspend in 0.1 M, pH 8.0 Tris buffer containing 2% BSA to 1 mg / mL to obtain HDV antigen-coated magnetic beads, aliquot and store at 4℃ for later use.

[0145] 2. Preparation of HDV polyclonal antibody-labeled acridine ester: Take 50 μL of 25 mg / mL HDV polyclonal antibody, add 150 μL of 0.1~0.2M pH 9.0~9.5 carbonate buffer, and mix well; add 1~2 μL of 5 mg / mL acridine ester and mix well. React at room temperature in the dark. After 1~2 h, remove the sample and desalt it using a 2 mL Zeba centrifugal desalting column. Collect the HDV polyclonal antibody-labeled acridine ester, aliquot it, and store at 4℃ for later use. This example uses the rabbit HDV polyclonal antibody prepared in Example 2 as an example.

[0146] 3. Preparation of the anti-HDV total antibody chemiluminescence detection kit:

[0147] (1) Prepare magnetic bead diluent with the following formula: 50mM Tris, 0.9% NaCl, 0.5% BSA, 0.05% Tween-20, 0.1% ProClin300, pH 7.5;

[0148] (2) Prepare acridine dilution solution with the following formula: 50 mM PB, 0.9% NaCl, 1% BSA, 0.5% Triton X-405, 0.1% ProClin300, pH 7.5;

[0149] (3) Prepare acridine working solution by diluting HDV polyclonal antibody-labeled acridine ester with acridine diluent; prepare magnetic bead working solution by diluting HDV antigen-coated magnetic nanobeads with magnetic bead diluent; assemble into an anti-HDV total antibody chemiluminescence detection kit.

[0150] 4. The methodology of this embodiment is a competition method, and the procedure is a two-step method:

[0151] (1) Step 1: Add 50 μL of 0.1 mg / mL HDV antigen-coated magnetic beads and 50 μL of the sample to be tested to the reaction vessel and incubate for 10 min; the anti-HDV antibody in the sample binds to the HDV recombinant antigen on the magnetic beads to form an antigen-antibody complex;

[0152] During cleaning, under the influence of a magnetic field, the magnetic beads are adsorbed onto the reaction tube wall, and the unbound substances are washed away.

[0153] (2) Second step: Add 50 μL of 0.1 μg / mL HDV polyclonal antibody-labeled acridine ester; incubate for 10 min; HDV polyclonal antibody-labeled acridine ester binds to the remaining sites of the recombinant antigen to form a magnetic bead-coated antigen-acidine-labeled antibody complex;

[0154] Wash again;

[0155] Excitation and reading: The chemiluminescent reaction was measured by relative luminescence intensity (RLU) after adding pre-excitation and excitation solutions to the reaction complex.

[0156] 5. Constructing a standard curve: Using the method described in step 4 above, detect samples with known concentrations and construct a standard curve between relative luminescence intensity (RLU) and the total amount of HDV antibody. The results are as follows: Figure 2 As shown, the results indicate that the standard curve fit is 0.999069, the LOD (limit of detection) is 0.136, and the COI (Cut-Off Index, a semi-quantitative or qualitative unit representing the ratio of the sample detection signal to the cut-off value) is negatively correlated with the total amount of anti-HDV antibodies in the sample and RLU.

[0157] Comparative Example 1

[0158] Total antibody determination using chemiluminescent double antibody sandwich method: Detection procedure: Magnetic beads are coated with the recombinant antigen HDV-SE prepared in Example 1 and acridine-labeled recombinant antigen, which bind to the HDV antibody in the sample to form a solid-phase antigen-antibody-signal antigen immune complex; excitation and reading are then performed.

[0159] Comparative Example 2

[0160] Chemiluminescence indirect method for determination of total antibody

[0161] Detection procedure: The recombinant antigen HDV-SE prepared in Biotinylated Example 1 binds to the HDV antibody in the sample to form an antigen-antibody immune complex. Then, SA magnetic beads are added to capture the immune complex, and then acrid ester is added to label the mouse anti-human IgG antibody; stimulation and reading are performed.

[0162] Example of effect 1

[0163] Characterization and performance data of the products from Example 4 and Comparative Examples 1 and 2 were compared:

[0164] The results of testing 35 clinically positive samples and 28 clinically negative samples using the products from Example 4 and Comparative Examples 1 and 2 are shown in Tables 1 and 2, respectively. The experimental results indicate that:

[0165] 1. Comparative Example 1: Both the double-antigen sandwich method and the indirect method of the anti-HDV total antibody detection kit have the risk of being misjudged as weakly positive due to the "hook effect" of high-concentration samples, and there is a risk of missed detection for extremely high-concentration samples. Furthermore, the double-antigen sandwich method has the problem of missed detection for some low-value samples due to the limited epitope coverage of recombinant antigen raw materials. The indirect method has the risk of false positives due to uneven background interference from IgG.

[0166] 2. The chemiluminescent competitive assay kit for total anti-HDV antibodies in Example 4, with its absolute resistance to the hook effect, ensures no false negatives even in high-concentration samples, providing the highest detection reliability and effectively preventing missed diagnoses. This is especially important for high-risk groups for hepatitis D virus (HDV) infection (such as patients with chronic hepatitis B) or patients with advanced liver disease. In these individuals, HDV antibody levels may be very high, and using sandwich or indirect methods poses a significant risk of the "hook effect," leading to missed diagnoses.

[0167] The anti-HDV total antibody detection kit in Example 3 uses a competitive mechanism, which can accurately report positive results, with uniform background, high sensitivity, and strong specificity. In the detection of the 63 clinical samples, there were no missed detections or false positives compared with nucleic acid.

[0168] Table 1. Detection results of three different methodologies and reagents in clinically positive HDV nucleic acid samples.

[0169]

[0170] Note: The concentration unit is COI; COI ≥ 1.00 is positive, and COI < 1.00 is negative.

[0171] Table 2. Detection results of three different methodological reagents on clinically negative samples for HDV nucleic acid testing.

[0172]

[0173] Note: The concentration unit is COI; COI ≥ 1.00 is positive, and COI < 1.00 is negative.

[0174] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0175] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims, and the specification and drawings can be used to interpret the content of the claims.

Claims

1. A method for preparing HDV polyclonal antibodies, characterized in that, This includes using HDV recombinant protein as an immunogen to immunize non-human animals and isolating polyclonal antibodies produced by the non-human animals; The HDV recombinant protein contains the following structure: From N-terminus to C-terminus: [S-HDAg]-[P1]-[P2]-[P3]; S-HDAg is a small antigen of HDV; P1 consists of amino acid residues 59 through 84 of the HDV small antigen. P2 consists of amino acid residues from position 92 to position 114 of the HDV small antigen. P3 consists of amino acid residues 149 to 172 of the HDV small antigen; The position of the amino acid residue is determined with reference to the amino acid sequence shown in SEQ ID NO.1, following the direction from the N-terminus to the C-terminus, with the first amino acid residue at the N-terminus of the amino acid sequence shown in SEQ ID NO.1 as the first position.

2. The method for preparing HDV polyclonal antibody according to claim 1, characterized in that, The amino acid sequence of the HDV small antigen is the amino acid sequence shown in SEQ ID NO.1, or an amino acid sequence that has at least 90% identity with the amino acid sequence shown in SEQ ID NO.1; Optionally, the amino acid sequence of P1 is the amino acid sequence shown in SEQ ID NO.2, or an amino acid sequence that has at least 90% identity with the amino acid sequence shown in SEQ ID NO.2; Optionally, the amino acid sequence of P2 is the amino acid sequence shown in SEQ ID NO.3, or an amino acid sequence that has at least 90% identity with the amino acid sequence shown in SEQ ID NO.3; Optionally, the amino acid sequence of P3 is the amino acid sequence shown in SEQ ID NO.4, or an amino acid sequence that has at least 90% identity with the amino acid sequence shown in SEQ ID NO.

4.

3. The method for preparing HDV polyclonal antibody according to claim 2, characterized in that, The HDV recombinant protein is directly or indirectly connected between any two domains; Optionally, the HDV recombinant protein further contains a tag; Optionally, the HDV recombinant protein is expressed using a prokaryotic system; more preferably, the HDV recombinant protein is expressed using an Escherichia coli expression system.

4. The method for preparing HDV polyclonal antibody according to claim 3, characterized in that, The amino acid sequence of the HDV recombinant protein is shown in SEQ ID NO.

5.

5. The method for preparing HDV polyclonal antibody according to any one of claims 1 to 4, characterized in that, The preparation method further includes collecting serum from non-human animals immunized with the recombinant HDV protein, and then purifying it to obtain the HDV polyclonal antibody.

6. The HDV polyclonal antibody prepared by the method for preparing HDV polyclonal antibodies according to any one of claims 1 to 5.

7. The use of the HDV polyclonal antibody according to claim 6 in any one of (i) to (v): (i) Detection of total anti-HDV antibodies; (ii) Prepare a kit for detecting total anti-HDV antibodies; (iii) Detection, purification, or enrichment of HDV antigen; (iv) Prepare kits for detecting, purifying or enriching HDV antigens; (v) Prepare a diagnostic kit for HDV infection.

8. A competitive assay kit for detecting total anti-HDV antibodies, characterized in that, It comprises (i) the recombinant HDV protein as defined in claim 1 or 2; and (ii) the HDV polyclonal antibody as described in claim 6.

9. The anti-HDV total antibody detection kit based on the competitive method according to claim 8, characterized in that, The HDV recombinant protein is linked to a solid-phase carrier, and the HDV polyclonal antibody is labeled with a signaling molecule. Optionally, the HDV polyclonal antibody is labeled with a chemiluminescent reagent; Optionally, the competitive method-based anti-HDV total antibody detection kit is used for chemiluminescent immunoassay and includes magnetic microparticles coated with the HDV recombinant protein, the HDV polyclonal antibody labeled with acridine ester, magnetic microparticle diluent, and acridine diluent.

10. A method for detecting total anti-HDV antibodies based on a competitive method, characterized in that, The method includes using the HDV recombinant protein as defined in claim 1 or 2 to bind to the total anti-HDV antibody in the sample, then using the HDV polyclonal antibody as described in claim 6 to bind to the vacant epitope of the HDV recombinant protein in the reaction system, and then obtaining the signal intensity of the signaling substance linked to the HDV polyclonal antibody, wherein the signal intensity is negatively correlated with the content of the total anti-HDV antibody. Optionally, the HDV recombinant protein is linked to a solid-phase support, and the HDV polyclonal antibody is labeled with a signaling molecule; Optionally, the HDV polyclonal antibody is labeled with a chemiluminescent reagent; Optionally, the HDV recombinant protein is linked to magnetic microparticles, and the HDV polyclonal antibody is labeled with acridine ester.

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