Antibodies against swine pseudorabies virus glycoprotein gB and uses thereof

Abstract

The application belongs to the technical field of immunology detection, and particularly relates to an antibody against porcine pseudorabies virus glycoprotein gB and application thereof. The antibody is a first antibody or a second antibody, the amino acid sequences of light chain CDR1-3 of the first antibody are respectively shown as SEQ ID NO. 3-5, the amino acid sequences of heavy chain CDR1-3 are respectively shown as SEQ ID NO. 8-10; the amino acid sequences of light chain CDR1-3 of the second antibody are respectively shown as SEQ ID NO. 13-15, and the amino acid sequences of heavy chain CDR1-3 are respectively shown as SEQ ID NO. 18-20. The antibody has good reactivity, recognition specificity and binding sensitivity with the PRV glycoprotein gB, is conducive to improving the detection sensitivity and accuracy of the gB protein or the anti-gB protein antibody, and provides an antibody tool with excellent performance for qualitatively or quantitatively evaluating the vaccine immunization effect of a pig group and detecting the content of porcine pseudorabies virus antigens in a sample.

Description

Technical Field

[0001] This invention relates to the field of immunological detection technology, and in particular to antibodies against porcine pseudorabies virus glycoprotein gB and their applications. Background Technology

[0002] Pseudorabies (PR) is an acute infectious disease caused by pseudorabies virus (PRV) in various animals, including pigs, cattle, sheep, dogs, cats, rabbits, rodents, wild boars, minks, bears, and foxes. The main clinical symptoms are fever, intense itching (except in pigs), and encephalomyelitis. Pseudorabies virus, also known as porcine herpesvirus type I, infectious bulbar paralysis virus, inflamed disease virus, and Oyetzki virus, belongs to the genus Varicellavirus of the subfamily C1 of the family Herpesviridae. It is a linear double-stranded DNA virus with a total gene length of approximately 150 kb, containing 77 reading frames, and an average G+C content as high as 73.6%. Pseudorabies is widespread in pigs. Infection with PRV in pigs mainly causes abortion, stillbirth, mummified fetuses in pregnant sows, and infertility in breeding pigs. Piglets exhibit loss of appetite, persistent diarrhea, and neurological symptoms with high mortality rates. Finishing pigs also experience respiratory symptoms. Moreover, PRV infection is prone to latent infection, leading to lifelong viral carriage and intermittent viral shedding. In addition, PRV is easily activated under stress conditions, which can cause recurrent infection and viral shedding. This latent-activation cycle mechanism determines the high incidence and explosive spread of pseudorabies virus in pig herds, making it one of the primary diseases that endanger pig herds and causing huge economic losses to the global pig industry.

[0003] Vaccination is a crucial measure for the prevention and control of infectious diseases. Before and after pseudorabies vaccination, it is necessary to promptly test the level of neutralizing antibodies in the serum of pig herds. This serves as a basis for assessing the effectiveness of the vaccination, understanding whether the vaccine has achieved the expected immunization effect, and rationally scheduling booster vaccinations. Furthermore, neutralizing antibody testing can also monitor wild-type virus infection status or disease treatment progress, which is of great significance for disease control.

[0004] Glycoprotein gB is a major immunogen of PRV (Porcine Rhinovirus) and plays a crucial role in the invasion phase of PRV replication. It facilitates the fusion of the cell membrane and viral envelope, enabling successful viral invasion, and mediates membrane fusion between infected and uninfected cells, aiding in viral transmission between cells. Vaccine development targeting gB protein has been very successful. gB protein can stimulate the body to produce complement-dependent and complement-independent neutralizing antibodies. Therefore, using PRV gB protein as a coating antigen, and competitively blocking the binding of serum neutralizing antibodies to gB protein with anti-gB protein antibodies, can efficiently evaluate immune antibody levels. This is a key step in the prevention and control of porcine PRV infection, the assessment of vaccine efficacy, and the eradication of porcine pseudorabies. Simultaneously, gB protein is also an important target in many current PRV virus detection kits. Therefore, the targeted development of monoclonal antibodies targeting glycoprotein gB is of great significance. Summary of the Invention

[0005] To address the technical problems existing in the prior art, this invention provides two antibodies with good reactivity, recognition specificity, and binding sensitivity against porcine pseudorabies virus glycoprotein gB, and antibody pairs thereof, along with the encoding genes and expression vectors of these antibodies. This invention further provides kits for preparing detection kits for porcine pseudorabies virus glycoprotein gB or anti-porcine pseudorabies virus glycoprotein gB antibodies, or the application of these antibodies in the preparation of anti-porcine pseudorabies virus drugs. This invention is specifically achieved through the following technical solutions:

[0006] The first aspect of this invention provides an antibody against porcine pseudorabies virus glycoprotein gB, selected from a first antibody or a second antibody, wherein the antibody comprises a light chain variable region and a heavy chain variable region, wherein: the amino acid sequences of the complementarity-determining regions CDR1-3 on the light chain variable region of the first antibody are shown in SEQ ID NO. 3-5, and the amino acid sequences of the complementarity-determining regions CDR1-3 on the heavy chain variable region are shown in SEQ ID NO. 8-10; the amino acid sequences of the complementarity-determining regions CDR1-3 on the light chain variable region of the second antibody are shown in SEQ ID NO. 13-15, and the amino acid sequences of the complementarity-determining regions CDR1-3 on the heavy chain variable region are shown in SEQ ID NO. 18-20.

[0007] Further, the amino acid sequence of the light chain variable region of the first 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; the amino acid sequence of the light chain variable region of the second antibody is shown in SEQ ID NO.12, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.17.

[0008] Further, the amino acid sequence of the first antibody light chain is shown in SEQ ID NO.1, and the amino acid sequence of the heavy chain is shown in SEQ ID NO.6; the amino acid sequence of the second antibody light chain is shown in SEQ ID NO.11, and the amino acid sequence of the heavy chain is shown in SEQ ID NO.16.

[0009] Further, the first antibody or the second antibody is a full-length antibody or its antigen-binding region; the antigen-binding region is selected from at least one of the Fab fragment, F(ab)2 fragment, Fv fragment, (Fv)2 fragment, scFv fragment and 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 an antibody against porcine pseudorabies virus glycoprotein gB as described above.

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

[0012] A fourth aspect of the present invention provides an antibody pair against porcine pseudorabies virus glycoprotein gB, comprising the first antibody and the second antibody as described above.

[0013] The fifth aspect of the present invention provides the use of the antibody, antibody-drug conjugate, or antibody pair against porcine pseudorabies virus glycoprotein gB as described above in the preparation of a kit for detecting porcine pseudorabies virus glycoprotein gB.

[0014] The sixth aspect of the present invention provides the use of the antibody, antibody-conjugate, or antibody pair against the pseudorabies virus glycoprotein gB as described above in the preparation of a kit for detecting the antibody against the pseudorabies virus glycoprotein gB.

[0015] The seventh aspect of the present invention provides the use of the antibody against porcine pseudorabies virus glycoprotein gB as described above in the preparation of anti-porcine pseudorabies virus drugs.

[0016] Compared with the prior art, the advantages and positive effects of the present invention are as follows:

[0017] 1. The antibody provided by this invention exhibits good reactivity, recognition specificity, and binding sensitivity to porcine pseudorabies virus glycoprotein gB. The immunoassay system constructed with this antibody for detecting gB protein neutralizing antibodies in porcine pseudorabies-positive serum has the advantages of good specificity, no cross-reactivity with other common viruses, and good linearity and a wide linear range in the detection results. This is beneficial for improving the detection sensitivity and accuracy of anti-gB protein antibodies and provides a high-performance antibody tool for qualitative or quantitative evaluation of the immunization effect of swine vaccines.

[0018] 2. The antibody provided by this invention recognizes different antigenic epitopes of gB protein and can be used as a pairing antibody. A double-antibody sandwich immunoassay system can be constructed by pairing the first antibody and the second antibody. The quantitative detection of gB protein has the characteristics of good specificity, wide linear range and high sensitivity. It has good application value in the fields of PVR virus content with gB protein as the detection target, identification of pseudorabies virus infected pigs and assistance in PVR purification.

[0019] 3. The antibody provided by this invention has the function of neutralizing PRV virus and blocking viral infection of cells by binding to gB protein, which is beneficial to providing a new means for the prevention and treatment of porcine pseudorabies and shows good clinical potential in the field of research and development of porcine pseudorabies antibody drugs. Detailed Implementation

[0020] 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.

[0021] 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.

[0022] 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.

[0023] 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.

[0024] 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.

[0025] The term “and / or” should be considered as a specific disclosure of each of the two specified features or components, with or without the other. For example, “A and / or B” is considered to include (i) A, (ii) B, and (iii) A and B.

[0026] The terms "rabbit monoclonal antibody," "monoclonal antibody," "rabbit-derived antibody," and "monoclonal antibody" have the same meaning. Unless otherwise specified, they all refer to antibodies that specifically bind to the porcine pseudorabies virus glycoprotein gB. The terms "glycoprotein gB," "gB protein," and "gB" have the same meaning. The modifier "rabbit" indicates that the antibody's complementarity-determining region (CDR) is derived from a rabbit immunoglobulin sequence.

[0027] 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.

[0028] 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 are relatively constant. The regions with significant amino acid sequence variation near the N-terminus in both the light and heavy chains 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 usually 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.

[0029] 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.

[0030] 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.

[0031] 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.

[0032] 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.

[0033] (i) Fab: An antigen-binding fragment (Fab) is a monovalent fragment consisting of a complete light chain (variable and constant regions) and a portion of a heavy chain (variable and first constant region). Fragments such as Fab, F(ab')2, and Fab' can be obtained by protease cleavage 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.

[0034] (ii)F(ab)2: Contains a bivalent segment consisting of two Fabs connected by a disulfide bridge in the hinge region.

[0035] (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.

[0036] (iv)(Fv)2: Consists of two Fv segments covalently linked together.

[0037] (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. The linker in this invention 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.

[0038] The (vi)sc(Fv)2 segment is formed by connecting two heavy chain variable regions and two light chain variable regions through a joint, etc.

[0039] 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.

[0040] 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.

[0041] 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.

[0042] 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.

[0043] Envelope glycoprotein gB is an essential glycoprotein for pseudorabies virus (PRV) replication. Both live vaccines and wild-type strains contain gB protein, and both live vaccine immunization and wild-type virus infection stimulate the body to produce gB antibodies. Therefore, collecting blood samples from immunized pigs, separating serum, and detecting gB protein-specific serum antibody levels are crucial for monitoring the effectiveness of pseudorabies vaccines and viral infection status. This is significant for immunization program development and monitoring of pseudorabies epidemic status. Enzyme-linked immunosorbent assay (ELISA) is the most commonly used method for PRV serological detection. Blocking gB-ELISA kits are often used to assess PRV antibody levels, monitoring the effectiveness of pig farm vaccinations and providing early warning of PRV wild-type virus infection. Currently, the relationship between the detected value of the commonly used ELISA method and the level of immune protection is unclear, making it difficult to promptly identify pigs with low levels of immune protection. When the number of these pigs reaches a certain level, the herd may become infected, leading to an increased carrier rate. Furthermore, the serum antibody detection values ​​of some kits are very similar, making it difficult to analyze their correspondence with neutralizing antibodies, thus hindering the accurate evaluation of the immunization efficacy of swine vaccines. Developing novel monoclonal antibodies targeting the glycoprotein gB to address these issues is of great significance.

[0044] This invention provides an antibody against porcine pseudorabies virus glycoprotein gB, which is either a first antibody or a second antibody. The 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. Specifically: the amino acid sequences of CDR1, CDR2, and CDR3 on the light chain variable region of the first antibody 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. Similarly, the amino acid sequences of CDR1, CDR2, and CDR3 on the light chain variable region of the second antibody are shown in SEQ ID NO. 13, SEQ ID NO. 14, and SEQ ID NO. 15, respectively; and the amino acid sequences of CDR1, CDR2, and CDR3 on the heavy chain variable region are shown in SEQ ID NO. 18, SEQ ID NO. 19, and SEQ ID NO. 10, respectively. Shown in NO.20.

[0045] This invention utilizes porcine pseudorabies virus (PRV) live vaccine to immunize New Zealand white rabbits, screening for two rabbit-derived monoclonal antibodies that exhibit good reactivity, recognition specificity, and binding sensitivity to the PRV glycoprotein gB. Immunoassay systems constructed using these antibodies, such as blocking ELISA and chemiluminescence assays, demonstrate high specificity and no cross-reactivity with positive sera from other common porcine viral diseases in detecting gB protein neutralizing antibodies in PRV-positive serum. The detection results show a good linear relationship with the neutralizing antibody concentration, with a linear range superior to commercially available kits of the same type. This allows for better differential detection of gB protein serum antibody levels, improving the sensitivity and accuracy of gB protein antibody detection and enabling accurate evaluation of the immunization effect of porcine vaccines. Furthermore, based on the different epitopes of the gB protein targeted by the antibodies in this invention, the two antibodies can be used as paired antibodies to construct a double-antibody sandwich immunoassay system. This system exhibits high specificity, a wide linear range, and high sensitivity in the quantitative detection of gB protein, showing significant application value in detecting PVR virus content targeting the gB protein, identifying PRV-infected pigs, and aiding in PRV eradication. Furthermore, the antibody of this invention also possesses the function of neutralizing PRV virus and blocking viral infection of cells by binding to the gB protein, and can be used for antiviral therapy. It also shows good clinical potential in the development of antibody drugs for porcine pseudorabies. In summary, the antibody against PRV glycoprotein gB provided by this invention has broad application prospects and important public health significance in the fields of PRV infection diagnosis, clinical treatment, and immune assessment.

[0046] 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 first antibody light chain variable region 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. The amino acid sequence of the second antibody light chain variable region is shown in SEQ ID NO.12, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.17.

[0047] Optionally, the 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.

[0048] Specifically, the amino acid sequence of the first 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. The amino acid sequence of the second antibody light chain (FL) is shown in SEQ ID NO.11, and the amino acid sequence of the heavy chain (FH) is shown in SEQ ID NO.16.

[0049] It should be noted that the 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.

[0050] 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 an antibody against the anti-porcine pseudorabies virus glycoprotein gB as described above.

[0051] 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.

[0052] 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.

[0053] 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.

[0054] 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.

[0055] 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, microinjection, electroporation, or liposome packaging 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 bombardment.

[0056] 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*), *Enterobacterium* strains (e.g., *Salmonella typhimurium*, *Serratia marcescens*), and *Pseudomonas* spp. Examples of 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 monoclonal antibodies.

[0057] Another embodiment of the present invention provides an antibody conjugate comprising an antibody against porcine pseudorabies virus glycoprotein gB as described above and a detection marker linked to the antibody.

[0058] It is important to emphasize that the monoclonal antibodies of this invention can be used alone or linked (covalently or non-covalently) with detection markers to form antibody-conjugates. The detection markers are used to directly or indirectly generate identifiable signal changes to identify the antibodies of this invention based on these signal changes, thereby qualitatively or quantitatively detecting the analyte through a specific antigen-antibody reaction. This includes, but is not limited to, antibodies against gB protein, PRV virus, and anti-gB protein. In some embodiments, the antibodies of this invention are used as antigen-binding (or capture) antibodies that specifically recognize and bind to gB protein in the sample to be tested. The analyte is then qualitatively or quantitatively detected by analyzing the signal from the detection marker linked to it. In other embodiments, the anti-gB protein antibody 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-gB protein antibody is a rabbit-derived IgG antibody, then the secondary antibody can be an anti-rabbit IgG antibody. Qualitative or quantitative detection of the analyte is achieved by analyzing the change in the detection marker signal generated after the secondary antibody specifically binds to the antibody of this invention. The antibodies of the present invention can also be used in combination, such as when used in combination, one of the two antibodies is used as a primary antibody or capture antibody, and the other is used as a secondary antibody or detection antibody.

[0059] 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.

[0060] In another embodiment of the present invention, an antibody pair against porcine pseudorabies virus glycoprotein gB is provided, which is composed of the first antibody and the second antibody as described above.

[0061] Based on the recognition of different gB protein epitopes by the antibodies of this invention, the first and second antibodies of this invention can be used as paired antibodies in a double antibody sandwich. Using these antibodies to construct a double antibody sandwich immunoassay method, such as a forensic luminescence detection system, can achieve specific and sensitive detection of gB protein, providing an effective approach for efficient and accurate detection of gB protein and detection of PRV virus targeting gB protein.

[0062] The present invention also provides the application of the antibody against porcine pseudorabies virus glycoprotein gB as described above, or its antibody conjugate or antibody pair thereof. Specifically, the present invention provides the following applications:

[0063] 1) The use of antibodies, antibody conjugates or antibody pairs against porcine pseudorabies virus glycoprotein gB as described above in the preparation of kits for detecting porcine pseudorabies virus glycoprotein gB;

[0064] 2) The use of antibodies, antibody conjugates or antibody pairs against porcine pseudorabies virus glycoprotein gB as described above in the preparation of kits for detecting antibodies against porcine pseudorabies virus glycoprotein gB;

[0065] 3) The application of the antibody against porcine pseudorabies virus glycoprotein gB as described above in the preparation of anti-porcine pseudorabies virus drugs.

[0066] 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.

[0067] When detecting gB protein, gB protein should be interpreted in the broadest sense, including not only pure gB protein but also mixtures containing gB protein or organisms containing gB protein such as PRV virus. In detecting gB protein, the antibody of the present invention is used to bind to the gB protein, and then the detection of gB protein is achieved by detecting the antibody of the present invention. Specifically, the gB 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 gB protein. For example, in a direct ELISA system, the antibody of the present invention is conjugated to a detection label, and the gB 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 gB antibody titer in the indirect ELISA detection system established in Example 1 of the present invention below. Of course, in other implementations, the analyte may not be coated. For example, in a double-antibody sandwich ELISA system, one of the antibodies of the present invention is coated onto a solid-phase carrier, and then the analyte binds to the antibody. Subsequently, the analyte is bound to another antibody conjugated with a detection label to achieve detection, as shown in the principle of the double-antibody sandwich ELISA detection system for measuring gB protein established in Example 5 of the present invention below. In a competitive ELISA system, the antibody of the present invention is coated onto a solid-phase carrier, and then the analyte conjugated with a detection label binds to the antibody to achieve detection. This is a conventional method in the art, and the present invention will not elaborate further.

[0068] When detecting gB protein antibodies, the antibody of this invention competitively binds to the gB protein antigen (produced by vaccine immunization) in the analyte, thereby enabling the detection of gB protein neutralizing antibody content. Specifically, a quantitative amount of gB protein is coated onto a solid-phase carrier, and then the antibody of this invention coupled with a detection label and the analyte simultaneously competitively bind to the coated antigen. The signal intensity generated by the detection label is inversely proportional to the level of gB protein neutralizing antibody in the analyte, thus the gB protein antibody content can be calculated based on the change in signal intensity, thereby assessing the immunization effect; this is the principle illustrated in the blocking ELISA established in Example 3 and the magnetic microparticle chemiluminescent immunoassay system established in Example 4 of this invention for determining gB neutralizing antibodies.

[0069] In the preparation of anti-swine pseudorabies virus drugs, the antibodies of this invention are used as the active pharmaceutical ingredient to neutralize the swine pseudorabies virus and achieve antiviral treatment.

[0070] 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.

[0071] Example 1: Preparation of monoclonal antibody against porcine pseudorabies virus glycoprotein gB

[0072] In this embodiment, New Zealand white rabbits were immunized with a commercially available live porcine pseudorabies virus (PRV) vaccine. Following this, B lymphocytes capable of recognizing the gB protein were enriched and screened from the spleen of the immunized rabbits using single-cell labeling and sorting technology. The isolated B lymphocytes were cultured as single cells to obtain secreted monoclonal antibodies. Finally, using recombinant gene expression technology, the 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 (CL) and heavy chain constant region (CH) genes, respectively, into expression vectors. These vectors were co-transfected into host cells, cultured, purified, and screened to obtain rabbit-derived monoclonal antibodies 17F12 and 20F3 against the gB protein. Antibody sequencing was performed by KingKin Biotechnology Co., Ltd. The amino acid (AA) sequences of the antibodies are shown in Table 1. In the table, LCDR1-3 represent the light chain complementarity-determining regions CDR1-3, and HCDR1-3 represent the heavy chain complementarity-determining regions CDR1-3, respectively.

[0073] Table 1. Sequence information of monoclonal antibodies 17F12 and 20F3 and immunogen in this embodiment.

[0074]

[0075]

[0076] 1.1 Animal Immunization: The immunogen used was a porcine pseudorabies heat-resistant protective live vaccine (Keqian Biotechnology; HB2000 strain). Each rabbit received an initial immunization of 0.4 doses, administered subcutaneously at multiple sites in the groin and back. Booster immunizations were given every two weeks after the initial immunization, with a dose of 0.2 doses, for a total of four booster immunizations. Ten days after the fifth immunization, blood was collected from the marginal ear vein to separate serum. The titer of PRV gB neutralizing antibodies in the serum was determined using an indirect enzyme-linked immunosorbent assay (ELISA) method coated with gB protein (amino acid sequence see SEQ ID NO.21). Rabbits with high serum titers received a booster immunization, and five days later, spleen cells were extracted from the sacrificed animals.

[0077] Preparation of gB protein: Based on the PRV-gB sequence shown in GeneBank accession number (MT949537.1), the gB gene was synthesized, and HindⅢ, XhoⅠ, and His tags were introduced upstream and downstream. The synthesized gB 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-gB 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 gB protein was obtained.

[0078] The indirect ELISA method for determining the titer of immune serum includes the following steps: (1) Coating: Prepare a 0.5 μg / mL PRV gB 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 Thermo Fisher, 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 pre-immunization rabbit serum as a negative control and the detection system without immune serum as a blank control (NC). See Example 3 for the detailed description of each buffer formulation.

[0079] The serum titer results are shown in Table 1. It can be seen that the titers of all three rabbits reached the required levels after five immunizations, indicating a strong immune response, which can be used for subsequent isolation of monoclonal antibodies.

[0080] Table 1. Results of serum anti-gB protein neutralizing antibody titers (OD) in three rabbits after five immunizations. 450 )

[0081] serum dilution Negative Rabbit No. 1 Rabbit No. 2 Rabbit No. 3 1:1000 0.314 1.679 1.712 1.698 1:3000 0.057 1.676 1.701 1.553 1:9000 0.036 1.5 1.582 1.317 1:27000 0.038 1.112 1.287 1.002 1:81000 0.03 0.585 0.697 0.463 1:243000 0.03 0.246 0.28 0.208 1:729000 0.029 0.092 0.106 0.071 1:2187000 0.029 0.045 0.066 0.038

[0082] 1.2 Isolation of B lymphocytes from the spleen and sorting and culture of antigen-specific B lymphocytes: For relevant methods, please refer to the published patents “Method for efficient isolation of 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)”.

[0083] The cultured B lymphocyte supernatant was used to identify positive clones that could recognize and bind PRVgB protein using an indirect ELISA coated with gB antigen. A total of 1152 cell supernatants were tested, and 7 clones (7A1, 2H5, 3G4, 8D8, 20F3, 17F12, and 5D5) with OD450 greater than 1.0 were identified as positive clones. The results are shown in Table 2.

[0084] Table 2 Results of antigen-specific B lymphocyte screening

[0085] Cloned ID 16E3 14B7 15H11 11A2 7H11 1A7 12H6 13A5 4E6 3F6 6B1 <![CDATA[OD 450 ]]> 0.323 0.355 0.357 0.369 0.369 0.372 0.372 0.384 0.401 0.404 0.524 Cloned ID 8H2 10C12 1G7 7A1 2H5 3G4 8D8 20F3 17F12 5D5 / <![CDATA[OD 450 ]]> 0.622 0.705 0.718 1.192 1.27 1.332 1.36 1.61 1.701 1.715 /

[0086] 1.3 Cloning of Rabbit Monoclonal Antibody Genes: B lymphocytes corresponding to positive clones were collected, lysed, and RNA was extracted and reverse transcribed into cDNA. Using the aforementioned cDNA as a template, the naturally paired rabbit antibody light chain variable region (VL) and heavy chain variable region (VH) genes were amplified by PCR. The PCR reaction system included: 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 included: 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.

[0087] The amplified DNA product was sequenced, and then the sequence of the constant region was obtained by querying the IMGT online database (www.imgt.org), resulting in antibody gene sequences of the intact light chain (FL) and the intact heavy chain (FH).

[0088] 1.4 Antibody Expression and Large-Scale Production: Rabbit monoclonal antibodies recognizing the PRVgB 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. The successful construction of the recombinant plasmid was verified by sequencing. The aforementioned process was outsourced to Wuhan Aibote Biotechnology Co., Ltd.

[0089] 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 gB protein recognition 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 manufacturer's manual. The antibody was then collected by dialysis and purified using 12% polyacrylamide gel electrophoresis (SDS-PAGE) to verify a purity ≥95%. After passing the verification, the antibody was aliquoted and stored at -20°C for later use.

[0090] Example 2: Neutralization experiment of monoclonal antibodies 17F12 and 20F3 against porcine pseudorabies virus

[0091] The neutralizing ability of antibodies 7A1, 2H5, 3G4, 8D8, 20F3, 17F12, and 5D5 against pseudorabies virus (PRV) was verified using the fixed virus-diluted antibody method. The steps included: (1) serially diluting the purified rabbit monoclonal antibody twice with cell maintenance medium, with an initial antibody concentration of 1 μg / mL and dilutions of 1:2, 1:8, 1:16, 1:32, 1:64, 1:128, and 1:256; (2) taking 50 μL of monoclonal antibody solutions of different dilutions and mixing them with 50 μL of standard pseudorabies virus (containing 100 half-maximal tissue cell infection doses (TCID50)). 50 (3) Mix the incubated monoclonal antibody and virus mixture and incubate it in a 37°C carbon dioxide incubator for 1 hour; (4) Transfer 100 μL of the incubated monoclonal antibody and virus mixture to a 96-well cell culture plate in which porcine kidney cells (PK-15) have grown into a monolayer. Inoculate 4 wells of cells for each dilution, and set up 4 wells of normal cell control and 4 wells of 100 TCID35. 50 For the virus control, a separate virus titer control group was set up. The virus was continuously diluted 10 times and added to a 96-well cell culture plate in which the cells had grown into a monolayer. Each dilution was inoculated into 4 wells, and 100 μL was added to each well. (4) The 96-well cell culture plate was placed in a 37°C, 5% CO2 cell culture incubator and the cytopathic effect (CPE) was observed daily. (6) Calculation of the 50% neutralization endpoint: According to the degree of cytopathic effect, the 50% neutralization endpoint was calculated using the Reed-Muench method. It is defined as the antibody dilution at which 50% of the cells do not produce a cytopathic effect (CPE).

[0092] The neutralizing ability of the antibodies of this invention against PRV is shown in Table 3. The results show that the screened antibodies 17F12 and 20F3 have good neutralizing effects against pseudorabies virus, with neutralizing antibody titers of 90.51 and 44.32, respectively, which can effectively neutralize pseudorabies virus.

[0093] Table 3. Results of the neutralizing ability test of the antibodies screened in this invention against PRV virus.

[0094]

[0095] Example 3: Analysis of the effectiveness of establishing a blocking enzyme-linked immunosorbent assay (ELISA) based on monoclonal antibody 17F12 for detecting gB neutralizing antibodies.

[0096] In this embodiment, gB protein is coated and immobilized on a solid-phase support. Then, the test serum and enzyme-labeled rabbit antibody 17F12 are added. The rabbit antibody 17F12 and the gB neutralizing antibody in the test serum competitively bind to the solid-phase antigen. Therefore, the amount of labeled antibody 17F12 bound to the solid phase is inversely proportional to the amount of gB neutralizing antibody in the test serum. Finally, a substrate is added for color development to detect the concentration of neutralizing antibody in the test serum.

[0097] The blocking ELISA system includes the following components: gB protein-coated microplate, washing buffer, dilution buffer (for diluting the sample to be tested), blocking buffer, enzyme-labeled monoclonal antibody 17F12, substrate solution, stop solution, and negative and positive controls for quality control. The washing buffer was PBS buffer (PBST buffer) containing 1% Tween-20, usually prepared as a 10-fold concentrated form. The dilution buffer and blocking buffer were phosphate buffer containing 0.5% (wt) casein (pH 7.4±0.1, concentration 0.2M). The substrate solution was 0.3 g / L TMB chromogenic solution. The stop solution was 2M H2SO4 solution. The negative control (NC) serum was diluted specific pathogen-free (SPF) grade experimental pig serum (purchased from Tianhang Biotechnology, catalog number 60011-8615), with an OD value of approximately 1.30. The positive control (PC) serum was diluted pig serum containing pseudorabies virus gB neutralizing antibody, with an OD value of approximately 0.20.

[0098] The preparation method of gB protein-coated ELISA plate includes: adding 0.25 μg / mL gB protein at 100 μL / well to a polystyrene microplate (96-well ELISA plate), 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.

[0099] The enzyme-labeled monoclonal antibody 17F12 was prepared using the 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).

[0100] 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 gB egg-coated ELISA plate 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 color development; measure the optical density (OD) value at 450 nm.

[0101] The criteria for determining the validity of the test are: the average positive control (PC) < 0.30 and the average negative control (NC) > 0.80. The S / N value is calculated as follows: S / N = average OD value of the sample to be tested / average OD value of the negative control; if S / N ≤ 0.4, the sample is considered positive for neutralizing antibodies; if S / N > 0.4, the sample is considered negative for neutralizing antibodies.

[0102] The linear range of the gB protein neutralizing antibody detection by blocking ELISA was validated. Porcine pseudorabies virus positive serum was diluted 2-, 4-, 8-, 16-, 32-, 64-, 128-, and 256-fold, respectively. The above detection system was then used in conjunction with the IDEXX pseudorabies virus gB protein antibody detection kit (catalog number 99-09732). The results are shown in Table 4. It can be seen that the linear range for detecting gB protein neutralizing antibodies in positive serum of this invention is between 16- and 256-fold dilutions, comparable to the linear range of commercially available kits. This provides an important antibody tool for the detection of gB protein neutralizing antibodies and the evaluation of the immunogenicity of positive serum containing these neutralizing antibodies.

[0103] Table 4. Linear range for detecting gB neutralizing antibodies using an enzyme-linked immunosorbent assay (ELISA) based on monoclonal antibody 17F12.

[0104] positive serum dilution factor Antibody 17F12 ELISA system (S / N) IDEXX Testing System (S / N) 2 0.0689(0.047) 0.0478(0.049) 4 0.0715(0.049) 0.0584(0.060) 8 0.0734(0.050) 0.0647(0.066) 16 0.0739(0.051) 0.0722(0.074) 32 0.183(0.126) 0.2113(0.217) 64 0.2162(0.149) 0.3056(0.313) 128 0.415(0.285) 0.3791(0.389) 256 0.6938(0.477) 0.5824(0.597) Mean of positive control 1.4557 0.9754 negative control mean 0.108 0.1634

[0105] The specificity of the neutralizing antibody blocking ELISA detection of gB protein was validated. The antigen coated was gB 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 5. The results showed that, except for pseudorabies virus positive swine serum with an S / N value ≤0.4 (classified as positive), the S / N values ​​of the other sera were between 0.7 and 0.9, and were all 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.

[0106] Table 5. Specificity of blocking ELISA detection of gB protein neutralizing antibodies

[0107]

[0108]

[0109] Example 4: Analysis of the effectiveness of establishing a magnetic microparticle chemiluminescence method for detecting gB neutralizing antibodies based on monoclonal antibody 17F12.

[0110] In this embodiment, gB protein is coated on the surface of magnetic microparticles, and then the test serum and acrid ester-labeled antibody 17F12 are added. The rabbit-derived antibody 17F12 and the gB neutralizing antibody in the test serum competitively bind to the solid-phase antigen to form a solid-phase coated antigen-labeled antibody complex. The amount of this complex is inversely proportional to the amount of gB neutralizing antibody in the test serum. 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 analyzer to detect the concentration of neutralizing antibody in the test serum.

[0111] The magnetic microparticle chemiluminescence system comprises the following components: gB protein-coated magnetic beads, washing buffer, acridinium ester-labeled monoclonal antibody 17F12, activation buffer, pre-activation buffer, and positive and negative calibrators for calibration, and quality control 1 and quality control 2 for quality control. The washing 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 1 are fetal bovine serum (purchased from Solarbio, catalog number S9030); and the positive calibrator and quality control 2 are 6 μg / mL gB monoclonal antibody 17F12.

[0112] The preparation method of gB 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, washing the magnetic beads three times with 100mM MES buffer (pH 5.0), adding 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysulfosuccinimide (sulfo-NHS) to a final concentration of 4mg / mL, reacting at room temperature for 30min, then washing the magnetic beads three times, adding gB protein (protein to magnetic beads mass ratio of 1:20), mixing well, and reacting at room temperature for 2h; finally washing the magnetic beads three times, adding preservation solution, and storing at 2-8℃.

[0113] The preparation method of acridine ester-labeled monoclonal antibody includes: taking 50 μL of antibody 17F12 (concentration 2 mg / mL) solution 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 2 h until the RLU of the peripheral dialysis buffer is less than 10000.

[0114] The magnetic microparticle chemiluminescence method includes the following steps: 20 μL of calibrator, quality control sample, or serum to be tested and 30 μL of gB protein-coated magnetic beads are added to a reaction vessel, mixed, and incubated at 37°C for 20 min. Unbound substances are washed away with washing buffer. Then, 30 μL of acridinium ester-labeled antibody 17F12 is added, mixed, and incubated at 37°C for 10 min. Unbound substances are washed away with washing buffer. Then, 50 μL of pre-activation solution and 50 μL of activation solution are added, and the reaction is carried out 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 value between 0.9 and 1.1 is considered valid; for quality control sample C2, an S / Co value between 0.04 and 0.06 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 value ≤ 0.7 is considered positive; an S / Co value ≥ 0.75 is considered negative; and a value between 0.7 and 0.75 is considered questionable.

[0116] The linear range of the gB protein neutralizing antibody detected by the magnetic particle chemiluminescence method was validated. Porcine pseudorabies virus positive serum was diluted 2-, 4-, 8-, 16-, 32-, 64-, 128-, and 256-fold, respectively. The detection system described above was used in conjunction with the IDEXX pseudorabies virus gB protein antibody detection kit. The results are shown in Table 6. It can be seen that the antibody of this invention exhibits a good trend in antibody titer change at positive serum dilutions from 4 to 258 times, while the imported ELISA kit does not show any titer change in serum before a 16-fold dilution. Therefore, the method for establishing the antibody of this invention has a wider linear range.

[0117] Table 6. Linear range for the detection of gB neutralizing antibodies using magnetic microparticle chemiluminescence immunoassay based on monoclonal antibody 17F12.

[0118] Standard positive serum dilution factor Antibody 17F12 magnetic microparticle chemiluminescence system (S / Co) IDEXX Testing System (S / N) 2 25624(0.031) 0.0478(0.049) 4 46397(0.056) 0.0584(0.060) 8 101818(0.123) 0.0647(0.066) 16 179529(0.217) 0.0722(0.074) 32 280856(0.339) 0.2113(0.217) 64 311524(0.376) 0.3056(0.313) 128 466428(0.563) 0.3791(0.389) 256 547886(0.661) 0.5824(0.597) Quality control product 1 533205 0.9754 Quality control product 2 828469 0.1634

[0119] The specificity of the magnetic particle chemiluminescence method for detecting gB protein neutralizing antibodies was validated. 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. The negative sera were confirmed negative for pseudorabies virus using an IDEXX ELISA kit. The results are shown in Table 7. The results showed that, except for the pseudorabies positive serum with an S / Co < 0.7, the S / Co values ​​of the other sera were all > 0.75, 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 7. Specificity of gB protein neutralizing antibody detection by magnetic particle chemiluminescence method.

[0121]

[0122] Example 5: Analysis of the effectiveness of establishing a magnetic microparticle chemiluminescence method for detecting gB protein content based on antibodies 17F12 and 20F3.

[0123] In this embodiment, a double-antibody sandwich method is used. Antibody 20F3 is coated on the surface of magnetic microparticles, and then the sample to be tested and acridinium ester-labeled antibody 17F12 are added sequentially to form an antibody 20F3-antigen-antibody 17F12 complex. Finally, pre-activation solution and activation solution are added. Acridinium ester absorbs the chemiluminescence energy in the chemical reaction and emits light. The PRVgB protein content in the sample is positively correlated with the luminescence value.

[0124] The magnetic microparticle chemiluminescence system comprises the following components: magnetic beads coated with monoclonal antibody 20F3, washing buffer, acridinium ester-labeled monoclonal antibody 17F12, activation buffer, pre-activation buffer, and positive and negative calibrators for calibration, and quality control 1 and quality control 2 for quality control. The negative calibrator and quality control 1 are in 0.01M PBS, while the positive calibrator and quality control 2 are gB proteins.

[0125] The preparation methods for monoclonal antibody 20F3 coated magnetic beads and acridine ester labeled monoclonal antibody 17F12 are described in Example 4.

[0126] The double-antibody sandwich magnetic microparticle chemiluminescence assay includes the following steps: Add 20 μL of calibrator, quality control, or test sample and 30 μL of monoclonal antibody 20F3-coated magnetic beads to a reaction vessel, mix well, and incubate at 37°C for 20 min. Wash away unbound material with washing buffer. Then add 30 μL of acridinium ester-labeled antibody 17F12, mix well, and incubate at 37°C for 10 min. Wash away unbound material with washing buffer. Add 50 μL of pre-activation buffer and 50 μL of activation buffer, react for 64 s, and immediately test the sample luminescence value using a fully automated chemiluminescence analyzer. The test sample contains serially diluted gB protein with concentrations of 100,000, 20,000, 4,000, 800, 160, 32, and 6.4 pg / mL.

[0127] Table 8 shows the luminescence values ​​of different concentrations of gB protein detected by the magnetic microparticle chemiluminescence method based on the antibody pair of the present invention. The standard curve obtained by linear fitting is: Y = 8.7677X + 6599, where X is the gB protein content, Y is the luminescence value, and RB is the luminescence value. 2 =0.998. The results show that the antibodies 17F12 and 20F3 of this invention can be used as paired antibodies for immunoassay. The established detection method has good dilution linearity and sensitivity, and can be used to calibrate the content of gB protein and the content of PRV virus containing gB protein.

[0128] Table 8. Standard curves for the detection of gB protein using magnetic microparticle chemiluminescence based on monoclonal antibodies 17F12 and 20F3.

[0129] gB protein concentration (unit: pg / mL) Luminous value 100000 878942 20000 205264 4000 36198 800 6923 160 1082 32 1069 6.4 1154 0 1125

[0130] 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. An antibody against porcine pseudorabies virus glycoprotein gB, characterized in that, Selected from a first antibody or a second antibody, said antibody includes a light chain variable region and a heavy chain variable region, wherein: The amino acid sequences of the complementarity-determining regions CDR1, CDR2 and CDR3 on the variable region of the first antibody light chain are shown in SEQ ID NO. 3-5, and the amino acid sequences of the complementarity-determining regions CDR1, CDR2 and CDR3 on the variable region of the heavy chain are shown in SEQ ID NO. 8-10, respectively. The amino acid sequences of the complementarity-determining regions CDR1, CDR2 and CDR3 on the variable region of the light chain of the second antibody are shown in SEQ ID NO. 13-15, and the amino acid sequences of the complementarity-determining regions CDR1, CDR2 and CDR3 on the variable region of the heavy chain are shown in SEQ ID NO. 18-20, respectively.

2. The antibody against porcine pseudorabies virus glycoprotein gB according to claim 1, characterized in that, The amino acid sequence of the light chain variable region of the first 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; The amino acid sequence of the light chain variable region of the second antibody is shown in SEQ ID NO.12, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.

17.

3. The antibody against porcine pseudorabies virus glycoprotein gB according to claim 2, characterized in that, The amino acid sequence of the light chain of the first antibody is shown in SEQ ID NO.1, and the amino acid sequence of the heavy chain is shown in SEQ ID NO.6; The amino acid sequence of the light chain of the second antibody is shown in SEQ ID NO.11, and the amino acid sequence of the heavy chain is shown in SEQ ID NO.

16.

4. The antibody against porcine pseudorabies virus glycoprotein gB according to claim 1, characterized in that, The first antibody or the second antibody is a full-length antibody or the antigen-binding region of the full-length antibody; the antigen-binding region is selected from Fab, F(ab)2, Fab', F(ab')2, Fv, (Fv)2, scFv or sc(Fv)2.

5. A nucleic acid molecule or recombinant vector, characterized in that, The recombinant vector contains the nucleic acid molecule, which encodes an antibody against porcine pseudorabies virus glycoprotein gB as described in any one of claims 1-4.

6. An antibody conjugate, characterized in that, It consists of an antibody against porcine pseudorabies virus glycoprotein gB as described in any one of claims 1-4 and a detection marker linked to the antibody.

7. An antibody pair against porcine pseudorabies virus glycoprotein gB, characterized in that, It consists of the first antibody and the second antibody as described in any one of claims 1-4.

8. The use of the antibody against porcine pseudorabies virus glycoprotein gB as described in any one of claims 1-4, the antibody conjugate as described in claim 6, or the antibody against porcine pseudorabies virus glycoprotein gB as described in claim 7 in the preparation of a kit for detecting porcine pseudorabies virus glycoprotein gB.

9. The use of the antibody against porcine pseudorabies virus glycoprotein gB as described in any one of claims 1-4, the antibody conjugate as described in claim 6, or the antibody against porcine pseudorabies virus glycoprotein gB as described in claim 7 in the preparation of a kit for detecting antibodies against porcine pseudorabies virus glycoprotein gB.

10. The use of the antibody against porcine pseudorabies virus glycoprotein gB as described in any one of claims 1-4 in the preparation of an anti-porcine pseudorabies virus drug.

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