Rabbit monoclonal antibody against pseudorabies virus glycoprotein gE and its application in immunodetection

Abstract

The application belongs to the technical field of immunology detection, and particularly relates to a rabbit monoclonal antibody against a pseudorabies virus glycoprotein gE and application thereof in immunological detection. The amino acid sequences of CDR1, CDR2 and CDR3 on the light chain variable region of the rabbit monoclonal antibody are respectively shown as SEQ ID NO. 3-5, and the amino acid sequences of CDR1, CDR2 and CDR3 on the heavy chain variable region are respectively shown as SEQ ID NO. 8-10. The application provides a rabbit monoclonal antibody against a PRV glycoprotein gE and an immunological detection system constructed by using the antibody, which provides an important technical means for rapid differential diagnosis and large-scale screening of wild-type PRV virus-infected pigs and vaccine-immunized pigs, has the advantages of strong specificity, high accuracy, good sensitivity, simple detection method operation and the like, has a wide application prospect in the field of PRV pathogen purification, and has important application and popularization values.

Description

Technical Field

[0001] This invention relates to the field of immunological detection technology, and in particular to a rabbit monoclonal antibody against pseudorabies virus glycoprotein gE and its application in immunological detection. Background Technology

[0002] Pseudorabies (PR), also known as Aujeszky's disease, is an acute infectious disease caused by the pseudorabies virus (PRV). Its main symptoms include fever, intense itching, and central nervous system disorders. Pigs are the primary source of infection and natural host for PRV. Healthy pigs can become infected through direct contact with infected or carrier pigs. Adult pigs are generally asymptomatic carriers but remain so for life, shedding the virus. Infected sows may experience abortion, stillbirth, weak piglets, or mummified fetuses. Infected newborn piglets exhibit fever, loss of appetite, persistent diarrhea, and neurological symptoms, and may even die from exhaustion. Once PRV infection develops, it is difficult to eradicate. Its high incidence and explosive spread have caused enormous economic losses to the global pig industry, making it one of the major infectious diseases seriously threatening the healthy development of the pig farming sector. Preventing and controlling the widespread transmission of this disease is a formidable task currently facing us.

[0003] Currently, the prevention and control of pseudorabies primarily relies on vaccination, combined with the detection of pseudorabies virus antigen and serum neutralizing antibodies to differentiate between immunized and naturally infected animals, and to cull and eradicate wild-type infected pigs. Therefore, monitoring and detecting the levels of PRV virus or neutralizing antibodies in pig serum is a crucial step in pseudorabies prevention and control. The surface envelope glycoprotein gE is an important virulence factor of the pseudorabies virus and a key target in many current PRV wild-type strain infection detection kits. Furthermore, commonly available PRV vaccines are gE gene-deleted vaccines. After immunization with gE gene-deleted vaccines, antibodies against the gE protein cannot be detected in the serum of immunized pigs, while specific antibodies against the gE protein (infection antibodies) can be detected in the serum of pigs infected with wild-type strains. Therefore, monitoring the level of anti-gE protein antibodies in a pig herd can directly reflect the status of PRV wild-type infection, allowing for the screening and timely culling of wild-type infected pigs, thereby establishing a healthy, negative herd.

[0004] Specific antibodies for detecting porcine PRV glycoprotein gE are mainly detected using the blocking enzyme-linked immunosorbent assay (ELISA) and the more accurate chemiluminescent immunoassay (CLIA). The key to establishing these methods lies in developing monoclonal antibodies that specifically bind to glycoprotein gE with moderate affinity. The suitability of antibody performance directly determines the sensitivity and specificity of the entire immunoassay system. If the antibody affinity is too high, positive serum may not effectively block the binding of the labeled antibody to the antigen, resulting in false negatives. If the antibody affinity is too low, the binding of positive serum to the antigen may not be sensitively detected, also potentially leading to false negatives. If the antibody specificity is poor, it may cross-react with other antigens, resulting in non-specific blocking of the sample and thus false positives. Therefore, screening for rabbit monoclonal antibodies against PRV glycoprotein gE with high specificity and moderate affinity has significant practical application value. Summary of the Invention

[0005] To address the technical problems existing in the prior art, this invention provides a rabbit monoclonal antibody with good reactivity and recognition specificity against pseudorabies virus glycoprotein gE, and provides the encoding gene and expression vector of this antibody. This invention further provides a kit for preparing antibodies for detecting pseudorabies virus glycoprotein gE or anti-pseudorabies virus glycoprotein gE, or the application of the aforementioned antibody in preparing anti-pseudorabies virus drugs and corresponding kits. This invention is specifically achieved through the following technical solutions:

[0006] The first aspect of this invention provides a rabbit monoclonal antibody against pseudorabies virus glycoprotein gE, the rabbit monoclonal antibody comprising a light chain variable region and a heavy chain variable region, wherein the amino acid sequences of CDR1, CDR2 and CDR3 on the light chain variable region are shown in SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.5, respectively, and the amino acid sequences of CDR1, CDR2 and CDR3 on the heavy chain variable region are shown in SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO.10, respectively.

[0007] Furthermore, the amino acid sequence of the light chain variable region of the rabbit monoclonal antibody is shown in SEQ ID NO.2, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.7.

[0008] Furthermore, the amino acid sequence of the light chain of the rabbit monoclonal antibody is shown in SEQ ID NO.1, and the amino acid sequence of the heavy chain is shown in SEQ ID NO.6.

[0009] Furthermore, the rabbit monoclonal antibody is a full-length antibody or its antigen-binding region; the antigen-binding region is selected from the Fab fragment, F(ab)2 fragment, Fv fragment, (Fv)2 fragment, scFv fragment, or sc(Fv)2 fragment.

[0010] A second aspect of the present invention provides a nucleic acid molecule or recombinant vector, the recombinant vector comprising the nucleic acid molecule, the nucleic acid molecule encoding a rabbit monoclonal antibody against pseudorabies virus glycoprotein gE as described above.

[0011] A third aspect of the present invention provides an antibody conjugate comprising a rabbit monoclonal antibody against pseudorabies virus glycoprotein gE as described above and a detection marker linked to the rabbit monoclonal antibody.

[0012] Furthermore, the detection marker is horseradish peroxidase or acridine ester.

[0013] The fourth aspect of the present invention provides the use of the rabbit monoclonal antibody or antibody-conjugate against pseudorabies virus glycoprotein gE as described above in the preparation of a detection kit for detecting pseudorabies virus glycoprotein gE or in the preparation of a detection kit for detecting an antibody against pseudorabies virus glycoprotein gE.

[0014] The fifth aspect of the present invention provides a detection kit comprising a rabbit monoclonal antibody or antibody-drug conjugate against pseudorabies virus glycoprotein gE as described above.

[0015] Compared with the prior art, the advantages and positive effects of the present invention are as follows:

[0016] This invention provides a rabbit monoclonal antibody with good reactivity and recognition specificity to PRV glycoprotein gE. When used to construct immunoassay systems such as blocking ELISA and chemiluminescent immunoassay systems, the reaction system can specifically detect gE antibodies in the serum of individuals infected with pseudorabies virus. It exhibits no cross-reactivity or non-specific binding reactions with positive sera from other common porcine infectious diseases, demonstrating advantages such as high specificity, high accuracy, good sensitivity, and simple operation. This invention provides a rabbit monoclonal antibody against PRV glycoprotein gE and a detection system constructed using this antibody, offering an important technical means for rapid differential diagnosis and large-scale screening of wild-type PRV virus-infected pigs and vaccinated pigs. It has broad application prospects in the field of PRV pathogen eradication and has significant application and promotion value. Detailed Implementation

[0017] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. The embodiments described herein are for illustrative purposes only and are not intended to limit the invention.

[0018] Based on the information contained herein, various changes to the precise description of the invention can be readily made by those skilled in the art without departing from the spirit and scope of the appended claims. It should be understood that the scope of the invention is not limited to the defined processes, properties, or components, as these embodiments and other descriptions are merely illustrative of specific aspects of the invention. In fact, various modifications to embodiments of the invention that will be apparent to those skilled in the art or related fields are covered within the scope of the appended claims.

[0019] To better understand the invention and not to limit its scope, all figures and other numerical values ​​used in this invention to indicate amounts, percentages, or other quantities should, in all cases, be understood to be modified by the word "approximately." Therefore, unless specifically stated otherwise, the numerical parameters listed in the specification and appended claims are approximate values ​​and may vary depending on the desired properties being sought. Each numerical parameter should at least be considered as obtained based on reported significant figures and through conventional rounding methods.

[0020] Additionally, it should be noted that, unless otherwise defined, the scientific and technical terms used in the context of this invention should have the meanings commonly understood by those skilled in the art.

[0021] The terms “including,” “contains,” “includes,” “has,” and similar words are non-restrictive and can include other steps and other components that do not affect the result.

[0022] The term “and / or” should be regarded as referring to a specific disclosure of each of the two specified features or components, with or without the other. For example, “A and / or B” is regarded as including (i) A, (ii) B, and (iii) A and B.

[0023] The terms "rabbit monoclonal antibody," "monoclonal antibody," "rabbit-derived antibody," and "monoclonal antibody" have the same meaning. Unless otherwise specified, they all refer to rabbit monoclonal antibodies that specifically bind to the pseudorabies virus glycoprotein gE. The terms "glycoprotein gE," "gE protein," and "gE" have the same meaning. The modifier "rabbit" indicates that the antibody's complementarity-determining region (CDR) is derived from a rabbit immunoglobulin sequence.

[0024] An antibody is an immunoglobulin molecule that specifically binds to a target antigen or epitope through at least one antigen recognition site located in the variable region of the immunoglobulin molecule. In this invention, the term "antibody" is to be interpreted in the broadest sense and includes various antibody structures, including but not limited to so-called full-length antibodies, antibody fragments, and their genetic or chemical modifications, provided they exhibit the desired antigen-binding activity. An antibody fragment may be one or more portions or fragments of a full-length antibody, retaining the antibody's ability to specifically bind to a target antigen.

[0025] A typical antibody molecule (full-length antibody) consists of two identical light chains (L) and two identical heavy chains (H). Light chains can be classified into two types: κ chains and λ chains; heavy chains can be classified into five types: μ, δ, γ, α, and ε chains, with antibodies defined as IgM, IgD, IgG, IgA, and IgE, respectively. The amino acid sequences near the N-terminus of both the heavy and light chains vary considerably, while the amino acid sequences of other parts remain relatively constant. The regions of the light and heavy chains with significant amino acid sequence variation near the N-terminus are called variable regions (V), and the regions with relatively stable amino acid sequences near the C-terminus are called constant regions (C). The variable regions of the heavy chain (VH) and light chain (VL) are typically the most variable parts of the antibody and contain antigen recognition sites. The VH and VL regions can be further subdivided into hypervariable regions (HVR) and framework regions (FR). The hypervariable region, also known as the complementarity-determining region (CDR), is a ring structure. Heavy chain CDRs and light chain CDRs are tightly joined together by the FR region and cooperate to form a surface that is complementary to the three-dimensional structure of the target antigen or epitope, determining the antibody's specificity and serving as the site for antibody recognition and antigen binding. The FR regions are the more conserved parts of the VH and VL, generally exhibiting a β-sheet configuration, linked by three CDRs forming a connecting loop. Each VH and VL typically consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0026] CDRs and FRs can be identified according to the Kabat definition, the Chothia definition, the sum of the Kabat and Chothia definitions, the AbM definition, the contact definition, the IMGT unique numbering definition and / or the conformation definition, or any CDR determination method known in the art.

[0027] The light chain constant region (CL) and heavy chain constant region (CH) do not directly participate in antibody-antigen binding, but they exhibit different effector functions, such as participating in antibody-dependent cell-mediated cytotoxicity (ADCC). The CL lengths of different Ig types (κ or λ) are generally consistent, but the CH lengths differ among Ig classes. For example, IgG, IgA, and IgD include CH1, CH2, and CH3, while IgM and IgE include CH1, CH2, CH3, and CH4. The amino acid sequences of the antibody heavy and light chain constant regions are well-known in the art and can be obtained by searching the IMGT database.

[0028] Full-length antibodies are the most complete antibody molecular structures, with a typical Y-type molecular structure. Therefore, in the context of this invention, "full-length antibody," "complete antibody," and "Y-type antibody" have the same meaning and can be used interchangeably.

[0029] An antibody fragment is one or more portions or segments of a full-length antibody that substantially retain the same biological function or activity as the full-length form. Specifically, an antibody fragment includes at least the same CDR region as the full-length antibody, and more preferably the same variable region, thereby retaining complete antigen recognition and binding sites, enabling it to bind to the same antigens, especially the same epitopes, as the full-length antibody. Typical examples of antibody fragments include Fab, F(ab)2, Fab', F(ab')2, Fv, (Fv)2, scFv, and sc(Fv)2, which can be obtained using conventional techniques in the art.

[0030] (i) Fab: An antigen-binding fragment (Fab) is a monovalent fragment consisting of a complete light chain (variable region and constant region) and a portion of a heavy chain (variable region and first constant region). Fragments such as Fab, F(ab')2, and Fab' can be obtained by protease digestion of a full-length antibody. For example, under the action of papain, IgG can be degraded into two Fab fragments and one Fc fragment; under the action of pepsin, IgG can be degraded into one F(ab')2 fragment and one pFc' fragment. The F(ab')2 fragment is further reduced to form two Fab' fragments. Because Fab possesses an antigen-binding region and a portion of a constant region, it not only has antibody-antigen affinity and excellent tissue penetration like scFv, but also has a more stable structure.

[0031] (ii) F(ab)2: contains a bivalent segment consisting of two Fabs connected by a disulfide bridge in the hinge region.

[0032] (iii) Fv: The variable fragment (Fv) is located at the N-terminus of the antibody Fab fragment. It contains only the variable region and consists of a variable region of one light chain and one heavy chain. It is a non-covalently bound dimer of VH and VL (VH-VL dimer). The three CDRs of each variable region interact to form an antigen-binding site on the surface of the VH-VL dimer, which has the ability to recognize and bind antigens, although the affinity is lower than that of the intact antibody.

[0033] (iv) (Fv)2: Consists of two Fv segments covalently linked together.

[0034] (v) scFv: A single-chain variable fragment (scFv) is an Fv fragment composed of a single polypeptide chain, consisting of a heavy chain variable region (VH) and a light chain variable region (VL) linked by a flexible linker (typically composed of 10-25 amino acids). It retains the original antibody's specificity for binding to the antigen. In this invention, the linker is not particularly limited as long as it does not interfere with the expression of the antibody variable regions linked to its two ends. Compared to full-length antibodies, scFv has a smaller molecular weight, thus exhibiting higher penetration and lower immune side effects.

[0035] (vi)sc(Fv)2 segment is formed by connecting two heavy chain variable regions and two light chain variable regions through a joint, etc.

[0036] In some embodiments, the full-length sequence of the antibody or antibody fragment of the present invention may include a complementarity-determining region (CDR) and a framework region (FR) derived from a rabbit immunoglobulin sequence. In other embodiments, the antibody may contain amino acid residues encoded by a non-rabbit immunoglobulin sequence, such as porcine-derived antibodies, chimeric antibodies, etc., to reduce the body's rejection response while maintaining the desired specificity and affinity. The term "chimeric antibody" refers to an antibody in which a portion is derived from a specific source or species, while the remainder is derived from a different source or species. For example, a porcine-rabbit chimeric antibody is formed by binding a variable region of a rabbit antibody with a constant region of a porcine antibody. The term "porcine-derived antibody" is a chimeric antibody containing the CDR region of a non-porcine antibody, such as a rabbit antibody, and a FR region derived from a porcine antibody. This framework sequence may be derived from the FR sequence of a single or multiple other porcine antibody variable regions. In some cases, porcine-derived antibodies may also be formed by binding a CDR region of a rabbit antibody with a FR region and a constant region derived from a porcine antibody sequence. In the present invention, the CDR region in the chimeric antibody or porcine-derived antibody is derived from a rabbit CDR region.

[0037] The terms "monoclonal antibody" or similar terms are used interchangeably and refer to a homogeneous group of antibodies, meaning that the individual antibodies constituting the group are identical except for a small number of naturally occurring mutations and / or post-translational modifications (e.g., isomerization, amidation). A "monoclonal antibody" is highly specific, exhibiting a single binding specificity and affinity for the same or substantially identical epitopes on an antigen. The modifier "monoclonal" indicates that the antibody is obtained from a substantially homogeneous group of antibodies and should not be interpreted as limiting the source or method of preparation of the antibody. This antibody can be prepared by a variety of methods, including but not limited to hybridoma, phage display, yeast display, recombinant DNA, single-cell screening, or single-cell sequencing.

[0038] The term “specific binding” is a well-known term in the art. A molecule exhibits “specific binding” if it reacts more frequently, more rapidly, for a longer duration, and / or with greater affinity for a particular target antigen or epitope than it reacts with other target antigens or epitopes. “Specific binding”, or “preferred binding”, does not necessarily require (although may include) exclusive binding.

[0039] To make the objectives, features, and advantages of this invention more apparent and understandable, the specific embodiments of this invention are described in detail below.

[0040] The surface envelope glycoprotein gE is an important virulence factor of pseudorabies virus (PRV), but it is not essential for viral replication. Deletion or silencing of the gE gene can significantly reduce PRV virulence without affecting immunogenicity. Therefore, currently used live vaccines are mainly gE gene-deleted vaccines. Since the vaccine strain does not contain gE, it cannot stimulate the body to produce corresponding neutralizing antibodies against gE (hereinafter referred to as gE antibodies). Wild-type strains, however, can stimulate the production of gE antibodies. Therefore, by collecting blood samples from immunized pigs and detecting the level of gE protein-specific serum antibodies, it is possible to distinguish between wild-type infected individuals and those immunized with gE-deleted vaccines, culling wild-type infected individuals, and gradually eradicating PRV. In large-scale pig screening, serological testing is the most economical and efficient method. While ELISA was previously the most commonly used method in veterinary testing, emerging chemiluminescence technology can achieve more accurate detection than ELISA. Therefore, developing rabbit monoclonal antibodies targeting the gE glycoprotein and establishing a complete systematic detection method is of great significance for the detection of gE antibodies in pigs.

[0041] This invention provides a rabbit monoclonal antibody against pseudorabies virus glycoprotein gE. The rabbit monoclonal antibody includes a light chain variable region and a heavy chain variable region. Both the light chain variable region and the heavy chain variable region include three complementarity-determining regions (CDRs), named CDR1, CDR2, and CDR3, respectively. The amino acid sequences of CDR1, CDR2, and CDR3 on the light chain variable region are shown in SEQ ID NO.3, SEQ ID NO.4, and SEQ ID NO.5, respectively, and the amino acid sequences of CDR1, CDR2, and CDR3 on the heavy chain variable region are shown in SEQ ID NO.8, SEQ ID NO.9, and SEQ ID NO.10, respectively.

[0042] This invention utilizes the pseudorabies virus (PRV) glycoprotein gE to immunize rabbits, screening for rabbit-derived monoclonal antibodies that exhibit good reactivity and recognition specificity to PRV glycoprotein gE. Immunoassay systems, such as blocking ELISA and chemiluminescent assays, are constructed using the aforementioned antibody-conjugated detection markers. These systems can specifically detect gE antibodies in the serum of individuals infected with PRV. No cross-reactivity or non-specific binding reactions are observed with positive sera from other common swine infectious diseases (swine fever, porcine reproductive and respiratory syndrome (PRRS), porcine circovirus disease, porcine parvovirus disease, and swine foot-and-mouth disease type O). The detection results are highly accurate and sensitive. The serum dilution required for detecting PRV-positive swine serum is higher than that of commercially available kits of the same type, making it suitable for detecting low-concentration samples. This invention provides a rabbit monoclonal antibody against PRV glycoprotein gE and a detection system constructed using this antibody, offering an important technical means for the differential diagnosis and large-scale screening of wild-type PRV-infected pigs and vaccinated pigs. It has advantages such as high specificity, high accuracy, good sensitivity, and simple operation, and has broad application prospects in the field of animal disease eradication.

[0043] Optionally, both the light chain variable region and the heavy chain variable region include four frame regions (FRs), which are arranged in an alternating sequence with three core parameters (CDRs) to form the variable region. The amino acid sequence of the light chain variable region of the rabbit monoclonal antibody is shown in SEQ ID NO.2, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.7.

[0044] Optionally, the rabbit monoclonal antibody of the present invention further includes a light chain constant region (CL) and a heavy chain constant region (CH), wherein CL and VL constitute the light chain, and CH and VH constitute the heavy chain. The constant regions of the antibody are typically obtained by searching the IMGT online database, for example: searching for rabbit-derived IgG gamma C reign to obtain CH, and searching for rabbit-derived IgG Kappa C reign to obtain CL.

[0045] Specifically, the amino acid sequence of the rabbit monoclonal antibody light chain (FL) is shown in SEQ ID NO.1, and the amino acid sequence of the heavy chain (FH) is shown in SEQ ID NO.6.

[0046] It should be noted that the monoclonal antibody of the present invention can be a full-length antibody (having a typical Y-shaped molecular structure) or the antigen-binding region of the full-length antibody; the antigen-binding region refers to a polypeptide that substantially retains the same biological function or activity as the full-length form. Specifically, the antigen-binding region includes the CDR region as described above, and more preferably has the variable region as described above, thereby retaining an intact antigen recognition and binding site, capable of binding to the same antigen as the full-length antibody, especially to the same epitope. Optionally, the antigen-binding region is selected from at least one of Fab, F(ab)2, Fab', F(ab')2, Fv, (Fv)2, scFv, and sc(Fv)2. These antigen-binding regions can be obtained by conventional techniques in the art.

[0047] Another embodiment of the present invention provides a nucleic acid molecule, a recombinant vector containing the nucleic acid molecule, or a host cell containing the nucleic acid molecule, wherein the nucleic acid molecule is used to encode a rabbit monoclonal antibody against pseudorabies virus glycoprotein gE as described above.

[0048] Nucleic acid molecules can be in the form of DNA (such as cDNA, genomic DNA, or synthetic DNA) or RNA (such as mRNA or synthetic RNA). DNA can be single-stranded or double-stranded, and can be a coding strand or a non-coding strand.

[0049] The sequence of a nucleic acid molecule can be derived from the antibody AA sequence using conventional methods such as codon coding rules. The full-length sequence of a nucleic acid molecule or its fragments can usually be obtained using PCR amplification, recombination, or artificial synthesis.

[0050] The original vector used to construct the recombinant vector can be any vector conventional in the art, as long as it can contain the nucleic acid molecule. Typical vectors include plasmids (such as pBR322, pUC series, pET series, pGEX series), viral vectors, bacteriophages (such as λgt4λB, λ-Charon, λΔz1, and M13), viscera, and mini-chromosomes. The vector can be a cloning vector (i.e., used to transfer nucleic acid molecules into a host and multiply them in host cells) or an expression vector (i.e., containing the necessary genetic elements to allow the nucleic acid molecule inserted into the vector to be expressed in the host cell). The nucleic acid molecule is inserted into a suitable vector to form a cloning vector or expression vector carrying the nucleic acid molecule, which is then introduced into a host cell and cultured under specific conditions to express and obtain an antibody. This is a well-known technique in the art and will not be described in detail here.

[0051] The nucleic acid molecules encoding the monoclonal antibodies FL and FH of this invention can be inserted into two vectors, which can be introduced into the same or different host cells. When the heavy and light chains are expressed in different host cells, each chain can be isolated from the host cell expressing it, and the isolated heavy and light chains can be mixed and incubated under suitable conditions to form antibodies. In other embodiments, the nucleic acid molecules encoding antibodies FL and FH can also be cloned into a single vector, with each nucleic acid sequence ligated downstream of a suitable promoter; for example, each nucleic acid sequence encoding the heavy and light chains can be operatively ligated to different promoters, or the nucleic acid sequences encoding the heavy and light chains can be operatively ligated to a single promoter, such that both the heavy and light chains can be expressed by the same promoter. The choice of expression vector / promoter depends on the type of host cell used to produce the antibodies.

[0052] Recombinant vector transfection or transformation into host cells is performed using conventional techniques. When the host is a prokaryote such as *E. coli*, competent cells capable of absorbing DNA are harvested after the exponential growth phase and treated with CaCl2 or MgCl2; alternatively, methods such as microinjection, electroporation, liposome packaging, or gene gun can be used. When the host is a eukaryote, the following DNA transfection methods can be used: calcium phosphate coprecipitation, microinjection, electroporation, liposome packaging, or gene gun.

[0053] The host cell can be a prokaryotic or eukaryotic cell. Examples of prokaryotic host cells that can be used in this invention include, but are not limited to, *Escherichia coli* (e.g., DH5α, JM109, BL21, W3110), *Bacillus* spp. (e.g., *Bacillus subtilis*, *Bacillus thuringiensis*), *Enterobacter* strains (e.g., *Salmonella typhimurium*, *Serratia marcescens*), and *Pseudomonas* spp. Eukaryotic host cells that can be used for transformation include, but are not limited to, yeast, insect cells, and animal cells, such as Drosophila S2 or Sf9 cells, mammalian CHO, CHO DG44, CHO-S, COS-7, 293 series cells, HepG2, Huh7, 3T3, RIN, MDCK, and HEK293 cell lines. After obtaining host cells transfected or transformed with the recombinant vector described above, they can be cultured under suitable conditions to express antibodies, which can then be isolated to obtain purified antibodies.

[0054] In a preferred embodiment, the recombinant vector described above is the expression vector pcDNA3.1, and the host cell is human kidney epithelial cells (293F cells).

[0055] Another embodiment of the present invention provides an antibody conjugate comprising a rabbit monoclonal antibody against pseudorabies virus glycoprotein gE as described above and a detection marker linked to the rabbit monoclonal antibody.

[0056] It is important to emphasize that the rabbit monoclonal antibody of the present invention can be used alone or linked (covalently or non-covalently) with a detection marker to form an antibody-conjugate. The detection marker is used to directly or indirectly generate a identifiable signal change to identify the monoclonal antibody of the present invention based on said signal change, thereby qualitatively or quantitatively detecting the analyte through a specific antigen-antibody reaction, including but not limited to gE protein, PRV virus, and antibodies against gE protein. In some embodiments, the antibody of the present invention is used as an antigen-binding (or capture) antibody that specifically recognizes and binds to gE protein in the sample to be tested, and then the analyte is qualitatively or quantitatively detected by analyzing the signal of the detection marker linked to it. In other embodiments, the antibody against gE protein is not labeled (as a primary antibody or capture antibody), but the detection marker is coupled to a secondary antibody (as a detection antibody) or other molecules that can bind to the primary antibody. For example, if the anti-gE protein antibody is a rabbit-derived IgG antibody, then the secondary antibody can be an anti-rabbit IgG antibody, thereby achieving qualitative or quantitative detection of the analyte by analyzing the change in the detection marker signal generated after the secondary antibody specifically binds to the antibody of the present invention.

[0057] Detection markers include, but are not limited to: biotin, fluorescent dyes (such as acridinium ester, umbelliferone, fluorescein, anthocyanin, fluorescein isothiocyanate (FITC), rhodamine, dichlorotriazineamine fluorescein, dansyl chloride), fluorescent proteins (such as isophycocyanin, phycoerythrin, PerCP and phycocyanin), enzymes (such as alkaline phosphatase, acid phosphatase, β-galactosidase, glucose oxidase, horseradish peroxidase, acetylcholinesterase, avidin), colloidal gold, colored magnetic beads, latex particles, radionuclides, detection antibodies or combinations thereof.

[0058] In the embodiments listed in this invention, the detection marker is horseradish peroxidase or acridine ester.

[0059] This invention provides applications of the rabbit monoclonal antibody or antibody-conjugate against pseudorabies virus glycoprotein gE as described above. Specifically, this invention provides the following applications:

[0060] 1) The application of the rabbit monoclonal antibody or antibody-conjugate against pseudorabies virus glycoprotein gE as described above in the preparation of a detection kit for detecting pseudorabies virus glycoprotein gE;

[0061] 2) The application of the rabbit monoclonal antibody or antibody-conjugate against pseudorabies virus glycoprotein gE as described above in the preparation of a detection kit for detecting antibodies against pseudorabies virus glycoprotein gE.

[0062] The detection methods described above employ conventional immunoassay techniques. Detection kits include, but are not limited to: enzyme-linked immunosorbent assay (ELISA) kits, enzyme-linked immunospot (ELISPOT) kits, immunohistochemistry (IHC) kits, immunofluorescence (IF) kits, Western blotting (WB) kits, flow cytometry (FC) kits, and chemiluminescent immunoassay kits. Samples for testing include, but are not limited to, serum, plasma, urine, cell culture medium, and tissue homogenates.

[0063] When detecting gE protein, gE protein should be interpreted in the broadest sense, including not only pure gE protein but also mixtures containing gE protein or organisms containing gE protein such as PRV virus. In detecting gE protein, the antibody of the present invention is used to bind to the gE protein, and then the detection of gE protein is achieved by detecting the antibody of the present invention. Specifically, the gE protein analyte can be coated onto a solid-phase carrier, and then the antibody of the present invention can be used to specifically recognize and bind to the solid-phase gE protein. For example, in a direct ELISA system, the antibody of the present invention is conjugated to a detection label, and the gE protein can be qualitatively or quantitatively detected by analyzing the signal of the detection label attached to it; in an indirect ELISA system, the antibody of the present invention is not labeled (as a primary antibody), but the detection label is conjugated to a secondary antibody that can bind to the antibody of the present invention, and the qualitative or quantitative detection is achieved by analyzing the detection label signal generated by the secondary antibody, as illustrated in the principle of determining the gE antibody titer in the indirect ELISA detection system established in Example 1 of the present invention below. In competitive ELISA systems, the antibody of this invention is coated onto a solid-phase carrier, and then the detection is achieved by binding the antibody with the detection label-conjugated gE protein. This is a conventional method in the field, and will not be described in detail here.

[0064] When detecting gE antibodies, the antibody of this invention competitively binds to the gE protein in the analyte, thereby detecting the content of gE antibodies in the analyte. Specifically, a quantitative amount of gE protein is coated onto a solid-phase carrier, and then the analyte and the antibody of this invention conjugated with a detection label are added sequentially. The gE antibody in the analyte blocks the binding of the antibody of this invention to the coated antigen. At this time, the signal intensity generated by the detection label is inversely proportional to the gE antibody level in the analyte, and the content of gE antibodies in the analyte can be calculated based on the change in signal intensity; this is the principle shown in the blocking ELISA established in Example 2 and the chemiluminescent immunoassay system established in Example 3 of this invention for the determination of gE antibodies.

[0065] This invention also provides a detection kit comprising a rabbit monoclonal antibody or antibody-drug conjugate against pseudorabies virus glycoprotein gE as described above.

[0066] Optionally, the detection kit further includes at least one of gE protein-coated solid-phase carrier, washing buffer, negative serum, and positive serum.

[0067] The present invention will be further illustrated below with reference to specific embodiments. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions, such as those described in *Molecular Cloning: A Laboratory Manual (Fourth Edition)* published by Cold Spring Harbor Laboratory, or generally under the conditions recommended by the manufacturer.

[0068] Example 1: Preparation of rabbit monoclonal antibody against pseudorabies virus glycoprotein gE

[0069] In this embodiment, New Zealand white rabbits were immunized with gE protein (amino acid sequence shown in SEQ ID NO. 11). Then, based on single B lymphocyte labeling and sorting technology, B lymphocytes capable of recognizing gE protein were enriched and screened from the immunized rabbit spleen. The isolated B lymphocytes were cultured as single cells to obtain secreted monoclonal antibodies. Finally, using recombinant gene expression technology, naturally paired antibody light chain (VL) and heavy chain variable region (VH) genes were first obtained from the monoclonal antibody-secreting B lymphocytes via PCR amplification. These genes were then inserted in tandem with the light chain constant region (CL) and heavy chain constant region (CH) into an expression vector, respectively. The vector was co-transfected into host cells, cultured, and purified to obtain rabbit-derived monoclonal antibodies against gE protein. Antibody sequencing was performed by Kinkai Biotechnology Co., Ltd.

[0070] 1.1 Animal Immunization

[0071] Preparation of gE protein: Based on the PRV-gE sequence (GeneBank accession number: NC_006151), the gE gene was synthesized, and Hind III, Xho I, and His tags were introduced upstream and downstream. The synthesized gE gene fragment was then ligated into the vector pcDNA3.1. The ligation product was transformed into DH5α competent cells, and the recombinant plasmid pcDNA3.1-gE was obtained after plasmid extraction and sequencing confirmation. This recombinant plasmid was transfected into CHO cells. Cell culture was harvested when cell viability was ≤60%. After cell lysis, the cells were centrifuged at 12000×g for 20 min to remove cell debris. An appropriate amount of supernatant was subjected to SDS-polyacrylamide gel electrophoresis. Simultaneously, the supernatant was filtered through a 0.45 μm filter and purified using a Ni column. After dialysis and medium replacement, the PRV gE protein was obtained.

[0072] Animal Immunization: Three New Zealand White rabbits were immunized with gE protein. For the first immunization, each rabbit received 500 μg of gE protein, which was emulsified by mixing gE protein with Freund's complete adjuvant at a 1:1 ratio and then injected subcutaneously at multiple sites on the back and abdomen. Booster immunizations were then performed every two weeks, with each rabbit receiving 200 μg of gE protein, which was emulsified by mixing gE protein with Freund's incomplete adjuvant at a 1:1 ratio and then injected subcutaneously at multiple sites on the back and abdomen. Three booster immunizations were performed. On the tenth day after the fourth immunization, blood was collected from the marginal ear vein to separate serum. Antibody titers in the rabbit serum were detected using indirect enzyme-linked immunosorbent assay (ELISA). Rabbits with high antibody titers received a third booster immunization. Four days later, the spleen was sacrificed, and spleen cells were separated.

[0073] The indirect ELISA method for determining the titer of immune serum includes the following steps: (1) Coating: Prepare a 0.5 μg / mL PRV gE protein solution with carbonate buffer (pH 9.6), add 100 μL / well to the microplate, and coat overnight at 4°C; (2) Blocking: Wash with 300 μL / well using washing buffer PBST, then add 200 μL / well using blocking buffer, and block at 37°C for 1 h; (3) Gradual dilution and addition of serum to be tested: Repeat the washing process in (2) to wash the well plate, then perform a gradient dilution of the serum to be tested, starting with a 1:1000 dilution and performing a three-fold gradient dilution, adding 100 μL / well of the serum dilution solution to the well plate, and incubating at room temperature for 1 h; (4) Secondary antibody incubation: Repeat the washing process in (2) to wash the well plate, then add 100 μL / well to the microplate, and incubate at room temperature for 1 h; Add 100 μL / well of horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG (purchased from ThermoFisher, catalog number 31460) diluted 1:5000 and incubate at room temperature in the dark for 1 h; (5) Terminate the reaction and develop color: Repeat the washing process in (2) to wash the plate, then add 100 μL / well of TMB colorimetric solution, react at 25°C in the dark for 15 min, and finally add 100 μL / well of termination buffer to terminate the reaction. Measure the absorbance at 450 nm. Use rabbit serum before immunization as a negative control and the detection system without immunized serum as a blank control (NC).

[0074] The serum titer test results are shown in Table 1. It can be seen that the titers of all three rabbits reached the required levels after the fourth immunization, indicating a strong immune response, which can be used for subsequent isolation of monoclonal antibodies.

[0075] Table 1. Results of gE antibody titer in serum of three rabbits after quadruple immunization (OD) 450 )

[0076]

[0077] 1.2 Isolation of B lymphocytes from the spleen and sorting and culture of antigen-specific B lymphocytes

[0078] For relevant methods, please refer to the published patents “Method for efficiently isolating single antigen-specific B lymphocytes from spleen cells (Publication No.: CN110016462A, Publication Date: 2019-07-16)” and “An in vitro culture system for B lymphocytes and its application (Publication No.: CN111518765A, Publication Date: 2020-08-11)”.

[0079] The supernatant of cultured B lymphocytes was used to identify positive clones capable of recognizing and binding PRV gE protein using an indirect ELISA method coated with gE protein. A total of 983 cell supernatants were tested, among which 8 OD cell lines (16D1, 15G5, 11A11, 15F12, 1C2, 3F5, 10D1, and 12F2) were identified. 450 The result was high, indicating a positive clone. The detection results for positive clones are shown in Table 2.

[0080] Table 2 Results of antigen-specific B lymphocyte screening

[0081]

[0082] 1.3 Cloning of the rabbit monoclonal antibody gene

[0083] B lymphocytes corresponding to positive clones were collected, lysed, and RNA was extracted and reverse transcribed into cDNA. Using the cDNA as a template, the light chain variable region (VL) and heavy chain variable region (VH) genes of naturally paired rabbit antibodies were amplified by PCR. The PCR reaction system consisted of: 4 μL cDNA, 1 μL forward primer (10 mM), 1 μL reverse primer (10 mM), 12.5 μL 2×Gloria HiFi (from Ibotek), and 6.5 μL H2O. The PCR amplification program consisted of: 98℃ pre-denaturation for 30 s, followed by 40 cycles of 95℃ for 15 s, 60℃ for 15 s, and 72℃ for 40 s, and finally holding at 72℃ for 5 min.

[0084] The amplified DNA product was sequenced, and then the sequence of the rabbit constant region was obtained by querying the IMGT online database (www.imgt.org), resulting in antibody gene sequences of the complete light chain (FL) and the complete heavy chain (FH).

[0085] 1.4 Expression and large-scale production of rabbit monoclonal antibodies

[0086] Rabbit monoclonal antibodies recognizing the PRV gE protein were produced on a large scale using recombinant expression technology. The heavy chain and light chain genes of the rabbit monoclonal antibody-positive clones selected in the above steps were loaded downstream of the signal peptide of the expression vector pcDNA3.1 via homologous recombination. Sequencing confirmed the successful construction of the recombinant plasmids. The aforementioned process was outsourced to Wuhan Aibote Biotechnology Co., Ltd.

[0087] The light and heavy chain recombinant expression vectors were co-transfected into 293F cells. After 72-96 hours of transfection, the cell supernatant was collected to obtain a rabbit monoclonal antibody containing the recombinant gE protein. The target antibody was purified from the cultured cell supernatant using Protein A affinity gel resin (purchased from Tiandi Renhe; catalog number: SA023100), following the instructions in the Protein A affinity gel resin manual. The antibody was then collected by dialysis and purified using 12% polyacrylamide gel electrophoresis (SDS-PAGE) to verify a purity ≥95%. After successful verification, the antibody was aliquoted and stored at -20°C for later use.

[0088] 1.5 Screening of rabbit monoclonal antibodies

[0089] Rabbit monoclonal antibodies suitable for blocking assays to detect gE antibodies in samples were screened using chemiluminescent immunoassay (CLIA). Detailed procedures are described in Example 3. The signal-to-noise ratio (SNR) was calculated, representing the ratio of negative to positive samples. The screening results for antibody strains 16D1, 15G5, 11A11, 15F12, 1C2, 3F5, 10D1, and 12F2 are shown in Table 3.

[0090] Table 3. Results of rabbit monoclonal antibody screening

[0091]

[0092] The results showed that rabbit monoclonal antibody 11A11 had the highest signal-to-noise ratio, so 11A11 was selected as the labeling antibody for the blocking method to detect gE antibody.

[0093] The amino acid (AA) sequences of antibody 11A11 and immunogen gE protein are shown in Table 4. In the table, VL represents the light chain variable region, LCDR1-3 represent the light chain complementarity-determining regions CDR1-3, VH represents the heavy chain variable region, and HCDR1-3 represent the heavy chain complementarity-determining regions CDR1-3.

[0094] Table 4 Sequence information of rabbit monoclonal antibody 11A11 and immunogen

[0095]

[0096] Example 2: Analysis of the effectiveness of establishing a blocking enzyme-linked immunosorbent assay (ELISA) for detecting gE antibodies based on rabbit monoclonal antibody 11A11.

[0097] This embodiment uses the blocking of rabbit monoclonal antibody 11A11 binding to the antigen to detect gE antibodies in the test serum. The principle is as follows: gE protein is coated and immobilized on a solid-phase carrier—an ELISA plate. Then, the test serum is added first, followed by the enzyme-labeled antibody 11A11. Antibody 11A11 and the gE antibody in the test serum competitively bind to the immobilized gE protein. The amount of labeled antibody 11A11 bound to the solid phase is inversely proportional to the amount of gE antibody in the test serum. Finally, the substrate catalyzed by the labeled enzyme is added for color development to detect the concentration of gE antibody in the test serum.

[0098] The blocking ELISA system includes the following components: gE protein-coated microplate, washing buffer, dilution buffer (for diluting the test samples), blocking buffer, enzyme-labeled monoclonal antibody 11A11, substrate solution, stop solution, and negative and positive serum controls for quality control. The washing buffer is PBS buffer (PBST buffer) containing 1% Tween-20, typically prepared as a 10-fold concentrated solution. The dilution and blocking buffers are phosphate buffer containing 0.5% (wt) casein (pH 7.4 ± 0.1, concentration 0.2 M). The substrate solution is 0.3 g / L TMB chromogenic solution. The stop solution is 2 M H2SO4 solution. The negative control (NC) serum is diluted specific pathogen-free (SPF) grade experimental porcine serum (purchased from Tianhang Biotechnology, catalog number 60011-8615), with an OD value of approximately 1.30. The positive control (PC) serum is diluted porcine serum containing pseudorabies virus gE antibody, with an OD value of approximately 0.20.

[0099] The preparation method of gE protein-coated ELISA plate includes: adding 0.25 µg / mL of gE protein to a polystyrene microplate (96-well ELISA plate) at 100 μL / well, incubating overnight at 4°C, then washing once with PBST at 150 μL / well, then adding blocking buffer, incubating overnight at 4°C to block unbound sites, then spin-drying and drying in a desiccant chamber, preferably sealed and stored in a desiccant-containing package.

[0100] The enzyme-labeled monoclonal antibody 11A11 was prepared using a horseradish peroxidase (HRP) rapid labeling kit (purchased from Huzhou Yingchuang Biotechnology Co., Ltd., catalog number HRP-L-100). In actual use, the antibody was appropriately diluted according to its titer for each batch. The diluent used was Tris-HCl buffer (pH 7.6) containing 1% (wt) bovine serum albumin (BSA).

[0101] The blocking ELISA method includes the following steps: (1) Serum treatment: Dilute the serum to be tested with an equal volume of dilution buffer, i.e., add 60 μL of dilution buffer to 60 μL of serum to be tested and mix well; (2) Serum loading: Add 100 μL of diluted serum to each well of the enzyme-labeled plate coated with gE protein and incubate at 37°C for 30 min. Set up 2 wells each for positive control serum and negative control serum; (3) Washing: Discard the liquid in the wells, add 300 μL of washing buffer to each well, wash 4 times, and pat dry; (4) Add enzyme-labeled monoclonal antibody: Add 100 μL of enzyme-labeled monoclonal antibody to each well and incubate at 37°C for 30 min; (5) Repeat step (3) to wash the plate; (6) Color development: Add 100 μL of TMB color development solution to each well and incubate at 37°C in the dark for 10 min. Then add 50 μL of stop solution to each well to stop the color development; measure the optical density (OD) value at 450 nm.

[0102] The criteria for determining the validity of the test are: a positive control mean (PC) < 0.30 and a negative control mean (NC) > 0.80. The S / N value is calculated as follows: S / N = average OD value of the test sample / average OD value of the negative control; a test sample S / N ≤ 0.6 is considered gE antibody positive, S / N ≥ 0.70 is considered gE antibody negative, and 0.6 < S / N < 0.7 is considered suspicious.

[0103] The linear range and sensitivity of the blocking ELISA detection of gE antibodies were validated. Porcine serum positive for pseudorabies virus was diluted 2-, 4-, 8-, 16-, 32-, 64-, 128-, and 256-fold, respectively. The above detection systems were then used in conjunction with the IDEXX pseudorabies virus gE antibody detection kit (catalog number 99-09836). The results are shown in Table 5, where "-" indicates negative and "+" indicates positive. It can be seen that the sensitivity of this invention in detecting positive serum containing gE protein antibodies is comparable to that of commercially available kits.

[0104] Table 5. Sensitivity of the enzyme-linked immunosorbent assay (ELISA) for detecting gE antibodies based on rabbit monoclonal antibody 11A11.

[0105]

[0106] The specificity of the antibody blocking ELISA detection of gE protein was validated. The antigen coated was gE protein. Positive and negative sera for classical swine fever, porcine reproductive and respiratory syndrome (PRRS), porcine circovirus disease, porcine parvovirus disease, foot-and-mouth disease type O, and pseudorabies virus were detected. Negative sera were confirmed negative for pseudorabies virus using an IDEXX ELISA kit. The results are shown in Table 6. The results showed that, except for pseudorabies virus positive swine serum with an S / N value ≤0.6 (classified as positive), the S / N values ​​of all other sera were greater than 0.7, and all were classified as negative. This indicates that the method established in this invention has good specificity and no cross-reactivity with positive sera from other pathogens.

[0107] Table 6. Specificity of gE antibody detection based on the blocking enzyme-linked immunosorbent assay (ELISA) established using rabbit monoclonal antibody 11A11.

[0108]

[0109] Example 3: Analysis of the effectiveness of a chemiluminescent immunoassay for detecting gE antibodies based on rabbit monoclonal antibody 11A11.

[0110] In this embodiment, gE protein is coated on the surface of a solid-phase support—magnetic microparticles. Then, the serum to be tested and acrid ester-labeled rabbit monoclonal antibody 11A11 are added sequentially. The gE antibody in the serum to be tested binds to the gE protein on the solid-phase support, blocking the binding of antibody 11A11. Therefore, the amount of labeled antibody bound to the solid-phase support is inversely proportional to the amount of gE antibody in the serum to be tested. Finally, pre-activation solution and activation solution are added. Acrid ester absorbs the chemiluminescence energy in the chemical reaction and emits light, which can be detected by the optical system of a chemiluminescence immunoassay analyzer to detect the concentration of gE antibody in the serum to be tested.

[0111] The magnetic microparticle chemiluminescent immunoassay (CLIA) system comprises the following components: gE protein-coated magnetic beads, wash buffer, acridil ester-labeled rabbit monoclonal antibody 11A11, activation buffer, pre-activation buffer, and positive and negative calibrators for calibration, and quality control samples C1 and C2 for quality control. The wash buffer is an aqueous solution (pH 7.2) containing 25 mM Tris, 150 mM NaCl, and 0.1% Tween 20. The pre-activation buffer is an aqueous solution containing 0.1% H₂O₂ and 0.1 M HCl. The activation buffer is an aqueous solution containing 0.2 M NaOH and 2% Triton X-100. The negative calibrator and quality control sample C1 are fetal bovine serum (purchased from Solarbio, catalog number S9030), and the positive calibrator and quality control sample C2 are 6 μg / mL gE monoclonal antibody 11A11.

[0112] The preparation method of gE protein coated magnetic beads includes: taking carboxyl magnetic beads (purchased from JSR, catalog number MS160) into a centrifuge tube, performing magnetic separation on a magnetic rack, removing the supernatant, washing the magnetic beads three times with 100 mM MES buffer (pH 5.0), resuspending the magnetic beads in 100 mM MES buffer (pH 5.0), adding 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysulfosuccinimide (sulfo-NHS) to a final concentration of 2.5 mg / mL, reacting at room temperature for 30 min, then washing the magnetic beads three times, resuspending the magnetic beads in 100 mM MES buffer (pH 5.0) containing 0.05% Tween 20, adding gE protein at a protein-to-magnetic-bead mass ratio of 1:20, mixing well, and reacting at room temperature for 2 h; finally washing the magnetic beads three times and diluting them to 0.5 μg / mL.

[0113] The preparation method of acridine ester-labeled monoclonal antibody includes: taking 50 μL of antibody solution (concentration 2 mg / mL) into a brown centrifuge tube, adding 35 μL of 0.02 M PBS, mixing well, then adding 15 μL of 5 mM acridine ester solution dissolved in dimethyl sulfoxide (DMSO), mixing well, briefly centrifuging, and labeling at 1500 rpm at room temperature for 2 h in the dark. After briefly centrifuging the acridine ester-labeled antibody, it is placed in a dialysis bag, using 0.02 M PBS as the dialysis buffer, changing the dialysis buffer every 4 h, and after 3 changes of the dialysis buffer, the labeled substance is recovered and diluted to 0.5 μg / mL.

[0114] The magnetic microparticle chemiluminescence method includes the following steps: 20 μL of positive serum and negative serum, along with 30 μL of gE protein-coated magnetic beads, are added to a reaction vessel, mixed, and incubated at 37°C for 20 min. Unbound material is washed away with washing buffer. Then, 30 μL of acridinium ester-labeled antibody 11A11 is added, mixed, and incubated at 37°C for 10 min. Unbound material is washed away with washing buffer. 100 μL of pre-activation solution and 100 μL of activation solution are added, and the reaction is allowed to proceed for 64 s. The sample luminescence value (RLU) is measured using a fully automated chemiluminescence analyzer.

[0115] The criteria for determining the validity of the test are as follows: For quality control sample C1, an S / Co ratio between 1.1 and 1.3 is considered valid; for quality control sample C2, an S / Co ratio between 0.1 and 0.13 is considered valid. The S / Co value is calculated as follows: S / Co = Sample luminescence value / Cutoff value. A test sample with an S / Co ratio ≤ 0.6 is considered positive; an S / Co ratio ≥ 0.65 is considered negative; and a ratio of 0.6 < S / Co < 0.65 is considered questionable.

[0116] The sensitivity of the magnetic particle chemiluminescence immunoassay for detecting gE antibodies was validated. Porcine serum positive for pseudorabies virus was diluted 2-, 4-, 8-, 16-, 32-, 64-, 128-, and 256-fold, respectively. The detection system described above was then used in conjunction with the IDEXX pseudorabies virus gE protein antibody detection kit. The results are shown in Table 7, where "-" indicates negative and "+" indicates positive. It can be seen that the detection system of this invention can still detect positive serum at a 128-fold dilution, and its sensitivity is three dilutions higher than that of imported ELISA kits.

[0117] Table 7. Sensitivity of the chemiluminescent immunoassay for detecting gE antibodies based on rabbit monoclonal antibody 11A11

[0118]

[0119] The specificity of the magnetic particle chemiluminescence immunoassay for detecting gE antibodies was validated. Positive and negative sera for classical swine fever (CSF), porcine reproductive and respiratory syndrome (PRRS), porcine circovirus disease (PCV2), porcine parvovirus disease (PCV2), porcine foot-and-mouth disease (FMD) type O, and pseudorabies virus were detected. The negative sera were confirmed negative for pseudorabies virus using an IDEXX ELISA kit. The results are shown in Table 8. The results showed that, except for pseudorabies-positive swine serum with an S / Co < 0.6, the S / Co values ​​of the other sera were all > 0.65, and all were judged as negative. This indicates that the detection system of the present invention has good specificity and no cross-reactivity with positive sera from other pathogens.

[0120] Table 8. Specificity of chemiluminescent immunoassay for gE antibody detection based on rabbit monoclonal antibody 11A11

[0121]

[0122] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A rabbit monoclonal antibody against pseudorabies virus glycoprotein gE, characterized in that, The rabbit monoclonal antibody includes a light chain variable region and a heavy chain variable region. The amino acid sequences of CDR1, CDR2 and CDR3 on the light chain variable region are shown in SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.5, respectively, and the amino acid sequences of CDR1, CDR2 and CDR3 on the heavy chain variable region are shown in SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO.10, respectively.

2. The rabbit monoclonal antibody against pseudorabies virus glycoprotein gE according to claim 1, characterized in that, The amino acid sequence of the light chain variable region of the rabbit monoclonal antibody is shown in SEQ ID NO.2, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.

7.

3. The rabbit monoclonal antibody against pseudorabies virus glycoprotein gE according to claim 2, characterized in that, The amino acid sequence of the light chain of the rabbit monoclonal antibody is shown in SEQ ID NO.1, and the amino acid sequence of the heavy chain is shown in SEQ ID NO.

6.

4. The rabbit monoclonal antibody against pseudorabies virus glycoprotein gE according to claim 1, characterized in that, The rabbit monoclonal antibody is a full-length antibody or its antigen-binding region; the antigen-binding region is selected from the Fab fragment, F(ab)2 fragment, Fv fragment, (Fv)2 fragment, scFv fragment or sc(Fv)2 fragment.

5. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes a rabbit monoclonal antibody against pseudorabies virus glycoprotein gE as described in any one of claims 1-4.

6. A recombinant vector, characterized in that, The recombinant vector comprises the nucleic acid molecule as described in claim 5.

7. An antibody conjugate, characterized in that, It consists of a rabbit monoclonal antibody against pseudorabies virus glycoprotein gE as described in any one of claims 1-4, and a detection marker linked to the rabbit monoclonal antibody.

8. The antibody conjugate according to claim 7, characterized in that, The detection marker is horseradish peroxidase or acridine ester.

9. The use of the rabbit monoclonal antibody against pseudorabies virus glycoprotein gE as described in any one of claims 1-4, or the antibody conjugate as described in any one of claims 7-8, in the preparation of a detection kit for detecting pseudorabies virus glycoprotein gE, or in the preparation of a detection kit for detecting an antibody against pseudorabies virus glycoprotein gE.

10. A test kit, characterized in that, The test kit comprises a rabbit monoclonal antibody against pseudorabies virus glycoprotein gE as described in any one of claims 1-4 or an antibody conjugate as described in any one of claims 7-8.

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