Antibody for porcine reproductive and respiratory syndrome virus and uses thereof

Abstract

This content discloses an antibody or antigen-binding fragment thereof that can bind to porcine reproductive and respiratory syndrome virus (PRRSV), and the use of such an antibody or antigen-binding fragment thereof in creating an immunoassay method or device for PRRSV detection.

Description

[Technical Field]

[0001] This disclosure relates to an antibody or an antigen-binding fragment thereof, and more specifically, to an antibody or an antigen-binding fragment thereof against porcine reproductive and respiratory syndrome virus and its use. [Previous Technology]

[0002] Porcine reproductive and respiratory syndrome (PRRS) is considered by many to be the most important disease in the global swine industry today. PRRS causes severe reproductive losses, as well as increased mortality associated with secondary infections, and is associated with reduced feed conversion ratios and decreased daily weight gain. Unfortunately, the virus that causes PRRS has proven difficult to control.

[0003] PRRS virus is an enveloped single-stranded RNA virus classified in the Arteriviridae family (Cavanaugh, 1997). It causes a widespread swine disease, first described in the United States in 1987 as a "mystery swine disease" (Hill, 1990). The disease manifests as respiratory illness in pigs of all age groups and causes mortality in some younger pigs as well as severe reproductive problems in breeding sows.

[0004] Infection with Porcine Reproductive and Respiratory Syndrome Virus (PRRSSV) is prevalent in most pig farms worldwide. PRRSV has been found to cause low survival rates and poor feed conversion ratios (FCR) in infected pigs, resulting in significant economic losses for the pig industry. Currently, there are some commercially available PRRSV detection kits. However, commercially available PRRSV detection kits typically have long testing times and require a large amount of reagents. Therefore, developing PRRSV detection kits that can detect PRRSV more conveniently and rapidly is of particular importance.

[0005] Cross-reference to related applications

[0006] This application claims priority to U.S. Provisional Patent Application No. 63 / 321,209, filed March 18, 2022, entitled “ANTIBODY FOR PORCINE REPRODUCTIVE AND RESPIRATORY SYNDROMVE VIRUS (PRRSV)”, which is incorporated herein by reference in its entirety.

[0007] Sequence List

[0008] This application is based on a sequence list submitted in XML format. The XML file contains a sequence list titled "2022A-154597-SequenceListing.xml", which was created on March 16, 2023, and has a size of 13,704 bytes. The sequence list contained in this XML file is part of this specification and is incorporated herein by reference in its entirety. [Summary of the Invention]

[0009] This disclosure relates to an antibody or an antigen-binding fragment thereof, and more specifically, to an antibody or an antigen-binding fragment thereof against porcine reproductive and respiratory syndrome virus and its use.

[0010] In a first aspect of this disclosure, an antibody or antigen-binding fragment thereof against porcine reproductive and respiratory syndrome virus (PRRSV) is provided, wherein the antibody or antigen-binding fragment thereof comprises: a heavy chain variant domain comprising: a first heavy chain complementarity-determining region (CDR-H1) containing sequence number 1, a second heavy chain complementarity-determining region (CDR-H2) containing sequence number 2, and a third heavy chain complementarity-determining region (CDR-H3) containing sequence number 3; and a light chain variant domain comprising: a first light chain complementarity-determining region (CDR-L1) containing sequence number 4, a second light chain complementarity-determining region (CDR-L2) containing a Lys-Ala-Ser sequence, and a third light chain complementarity-determining region (CDR-L3) containing sequence number 5.

[0011] In another embodiment of the first aspect, the heavy chain variant domain contains the sequence number 6.

[0012] In another embodiment of the first aspect, the light chain variant domain contains the sequence number 7.

[0013] In a second aspect of this disclosure, an isolated nucleic acid encoding an antibody or an antigen-binding fragment thereof as described in the first aspect of this disclosure is provided, wherein the isolated nucleic acid encoding the antibody or the antigen-binding fragment thereof comprises a first segment encoding the heavy chain variant domain and a second segment encoding the light chain variant domain.

[0014] In another embodiment of the second aspect, the first segment encoding the heavy chain variant domain further comprises a sequence numbered 8.

[0015] In another embodiment of the second aspect, the second segment encoding the light chain variant domain further comprises the sequence number 9.

[0016] In a third aspect of this disclosure, a carrier is provided that contains the isolated nucleic acid described in the second aspect of this disclosure.

[0017] In a fourth aspect of this disclosure, a host cell is provided, comprising a vector as described in a third aspect of this disclosure.

[0018] In a fifth aspect of this disclosure, a method is provided for manufacturing an antibody or antigen-binding fragment thereof against porcine reproductive and respiratory syndrome virus, comprising: (a) culturing a host cell as described in the fourth aspect of this disclosure under suitable conditions to express the antibody or antigen-binding fragment thereof; and (b) recovering the antibody or antigen-binding fragment thereof.

[0019] In another embodiment of the fifth aspect, the heavy chain variant domain contains the sequence number 6.

[0020] In another embodiment of the fifth aspect, the light chain variant domain contains the sequence number 7.

[0021] In a sixth aspect of this disclosure, a method for detecting porcine reproductive and respiratory syndrome virus includes: contacting a sample with an antibody or an antigen-binding fragment thereof as described in a first aspect of this disclosure.

[0022] In another embodiment of the sixth aspect, the heavy chain variant domain contains the sequence number 6.

[0023] In another embodiment of the sixth aspect, the light chain variant domain contains the sequence number 7.

[0024] In a seventh aspect of this disclosure, a biological field-effect transistor (Bio-FET) comprises: a transistor region; and a detection region, wherein the detection region includes a detection surface, the detection surface being functionalized for binding an antibody or antigen-binding fragment of porcine reproductive and respiratory syndrome virus (PRRSV), wherein the antibody or antigen-binding fragment comprises: a heavy chain variation domain comprising: a first heavy chain complementarity-determining region (CDR-H1) containing sequence number 1, a second heavy chain complementarity-determining region (CDR-H2) containing sequence number 2, and a third heavy chain complementarity-determining region (CDR-H3) containing sequence number 3; and a light chain variation domain comprising: a first light chain complementarity-determining region (CDR-L1) containing sequence number 4, a second light chain complementarity-determining region (CDR-L2) containing a Lys-Ala-Ser sequence, and a third light chain complementarity-determining region (CDR-L3) containing sequence number 5.

[0025] In another embodiment of the seventh aspect, the heavy chain variant domain contains the sequence number 6.

[0026] In another embodiment of the seventh aspect, the light chain variant domain contains the sequence number 7.

[0027] In an eighth aspect of this disclosure, a method for detecting porcine reproductive and respiratory syndrome virus (PRRSV) using a bio-field effect transistor (Bio-FET), the method comprising: (a) contacting a sample with an antibody or an antigen-binding fragment thereof immobilized on a detection surface of the bio-field effect transistor, wherein the antibody or antigen-binding fragment comprises: a heavy chain variant domain comprising: a first heavy chain complementarity-determining region (CDR-H1) containing sequence number 1, a second heavy chain complementarity-determining region (CDR-H2) containing sequence number 2, and a third heavy chain complementarity-determining region (CDR-H3) containing sequence number 3; and a light chain variant domain comprising: a first light chain complementarity-determining region (CDR-L1) containing sequence number 4, a second light chain complementarity-determining region (CDR-L2) containing a Lys-Ala-Ser sequence, and a third light chain complementarity-determining region (CDR-L3) containing sequence number 5; and (b) An electrical signal obtained from the bio-field effect transistor was analyzed.

[0028] In another embodiment of the eighth aspect, the heavy chain variant domain contains the sequence number 6.

[0029] In another embodiment of the eighth aspect, the light chain variant domain contains the sequence number 7.

[0030] In a ninth aspect of this disclosure, a composition for detecting porcine reproductive and respiratory syndrome virus (PRRSV) is provided, wherein the composition for detecting PRRSV comprises an antibody against PRRSV or an antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof comprises: a heavy chain variant domain comprising: a first heavy chain complementarity-determining region (CDR-H1) containing sequence number 1, a second heavy chain complementarity-determining region (CDR-H2) containing sequence number 2, and a third heavy chain complementarity-determining region (CDR-H3) containing sequence number 3; and a light chain variant domain comprising: a first light chain complementarity-determining region (CDR-L1) containing sequence number 4, a second light chain complementarity-determining region (CDR-L2) containing a Lys-Ala-Ser sequence, and a third light chain complementarity-determining region (CDR-L3) containing sequence number 5.

[0031] In another embodiment of the ninth aspect, the heavy chain variant domain contains the sequence number 6.

[0032] In another embodiment of the ninth aspect, the light chain variant domain contains the sequence number 7.

[0033] In another embodiment of the ninth aspect, the composition for detecting PRRSV may be a biochip device, or a detection kit including a biochip device (e.g., Bio-FET), or a detection kit for Western ink dot assay, chemiluminescent microparticle immunoassay (CMIA), chemiluminescent immunoassay (CLIA), lateral fluid immunoassay (LFIA), or enzyme-conjugated immunosorbent assay (ELISA).

[0034] In a tenth aspect of this disclosure, there is provided the use of an antibody or an antigen-binding fragment thereof in the preparation of a composition for detecting porcine reproductive and respiratory syndrome virus (PRRSV) in a test subject or a sample from a test subject, wherein the antibody or the antigen-binding fragment thereof comprises: a heavy chain variant domain comprising: a first heavy chain complementarity-determining region (CDR-H1) containing sequence number 1, a second heavy chain complementarity-determining region (CDR-H2) containing sequence number 2, and a third heavy chain complementarity-determining region (CDR-H3) containing sequence number 3; and a light chain variant domain comprising: a first light chain complementarity-determining region (CDR-L1) containing sequence number 4, a second light chain complementarity-determining region (CDR-L2) containing a Lys-Ala-Ser sequence, and a third light chain complementarity-determining region (CDR-L3) containing sequence number 5, wherein the test subject is a pig.

[0035] In another embodiment of the tenth aspect, the heavy chain variant domain contains the sequence number 6.

[0036] In another embodiment of the tenth aspect, the light chain variant domain contains the sequence number 7.

[0037] In another embodiment of the tenth aspect, the test subject is a pig.

[0038] In the eleventh aspect of this disclosure, a bio-field effect transistor (Bio-FET) is provided in the preparation of a composition for detecting porcine reproductive and respiratory syndrome virus (PRRSV) in a test subject or a sample from a test subject, wherein the Bio-FET comprises an antibody or an antigen-binding fragment thereof, including: a heavy chain variant domain comprising: a first heavy chain complementarity-determining region (CDR-H1) containing sequence number 1, a second heavy chain complementarity-determining region (CDR-H2) containing sequence number 2, and a third heavy chain complementarity-determining region (CDR-H3) containing sequence number 3; and a light chain variant domain comprising: a first light chain complementarity-determining region (CDR-L1) containing sequence number 4, a second light chain complementarity-determining region (CDR-L2) containing a Lys-Ala-Ser sequence, and a third light chain complementarity-determining region (CDR-L3) containing sequence number 5.

[0039] In another embodiment of the eleventh aspect, the test subject is a pig.

[0040] In another embodiment of the eleventh aspect, the heavy chain variant domain contains the sequence number 6.

[0041] In another embodiment of the eleventh aspect, the light chain variant domain contains the sequence number 7.

[0042] In another embodiment of the eleventh aspect, the antibody or its antigen-binding fragment is immobilized on the detection surface of the Bio-FET.

[0043] When the above-mentioned antibody or its antigen-binding fragment is used, the antibody or its antigen-binding fragment can specifically recognize PRRSV and has good sensitivity, thereby achieving excellent detection of PRRSV and thus being applied to various purposes, such as Western ink dot method, chemiluminescent microparticle immunoassay (CMIA), chemiluminescent immunoassay (CLIA), lateral fluid immunoassay (LFIA), or enzyme-conjugated immunosorbent assay (ELISA), or biochip device (e.g., Bio-FET).

Implementation Method

[0048] The following detailed description and accompanying drawings are merely descriptions of the present embodiments, but are not the only forms of implementing or utilizing the specific embodiments of the present invention. The embodiments cover features of multiple specific embodiments and method steps and their order for constructing and operating these specific embodiments. However, other specific embodiments may also be used to achieve the same or equivalent functions and order of steps.

[0049] Definition

[0050] For convenience, specific proper nouns used in this specification, embodiments, and appended claims are concentrated herein. Unless otherwise defined in this specification, the scientific and technical terms used herein have the same meaning as understood and commonly used by one of ordinary skill in the art to which this invention pertains. Furthermore, unless conflicting with the context, the singular form of a noun used herein includes its plural form, and vice versa. Specifically, in this specification and the claims, the singular form "a" (a and an) includes the plural reference value, unless otherwise indicated by the context. In addition, in this specification and the claims, the expressions "at least one" and "one or more" have the same meaning, both representing a total of one, two, three, or more.

[0051] The terms "first," "second," and "third" in the specification and accompanying drawings of this invention are used to distinguish different objects rather than to describe a specific order.

[0052] The term "antibody" is used in the broadest sense, specifically encompassing monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, as long as they exhibit the desired biological activity. "Antibody fragments" comprise a portion of a full-length antibody, typically its antigen-binding region or variant region. Examples of antibody fragments include Fab, Fab', F(ab')2, rIgG and Fv fragments, diabodies, linear antibodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments.

[0053] An "antibody fragment" comprises only a portion of a complete antibody, wherein said portion retains at least one function typically associated with it when it is present in the complete antibody, and may retain most or all of those functions. In one embodiment, the antibody fragment comprises the antigen-binding site of the complete antibody and thus retains the ability to bind antigens. In another embodiment, the antibody fragment, such as an antibody fragment comprising an Fc region, retains at least one biological function typically associated with said Fc region when it is present in the complete antibody, such as antibody half-life regulation, antibody-dependent cell-mediated cytotoxicity (ADCC) function, and complement binding. In one embodiment, the antibody fragment is a monovalent antibody whose half-life in vivo is substantially similar to that of a complete antibody. For example, such an antibody fragment may comprise an antigen-binding arm linked to an Fc sequence that provides stability of the antibody fragment in vivo. The antibody fragments of the present invention can be in different forms, including, for example, variable fragments (Fv), single-chain variable fragments (scFv), antigen-binding fragments (Fab), low IgG (rIgG), and bivalent antibody fragments (F(ab')2), as well as single-chain antibodies.

[0054] The term "variable region" or "variable domain" of an antibody refers to the amino-terminal domain of the heavy or light chain of the antibody. These domains are usually the most variable sites in the antibody and contain antigen-binding sites.

[0055] The term "variable" refers to the fact that certain parts of the variant domain have widely different sequences among different antibodies, contributing to the binding and specificity of each antibody to its specific antigen. However, variability is not uniformly distributed throughout the entire variant domain of an antibody. It is concentrated in three segments within the light and heavy chain variant regions, known as complementarity-determining regions (CDRs) or hypervariable regions. The more conserved portions of the variant domain are called framework regions (FRs). The natural heavy and light chain variant domains each contain four FR regions, which mostly adopt a β-lamellar configuration, linked by three CDRs in a loop, and in some cases, form part of a β-lamellar structure. The CDRs in each chain are clustered together by the FR region, forming the antibody antigen-binding site together with the CDRs of another chain (see Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md. (1991)). Constant domains do not directly participate in antibody-antigen binding but exhibit various effector functions, such as antibody involvement in antibody-dependent cellular toxicity.

[0056] As used herein, the term "complementarity determining region" (CDR) refers to a highly variable region of an antibody molecule that forms a surface complementary to the three-dimensional surface of the antigen it binds to. Moving from the N-terminus to the C-terminus, each antibody's heavy and light chains contain three CDRs (CDR1, CDR2, and CDR3). Therefore, there are a total of six CDRs, including three from the heavy chain variable region and three from the light chain variable region.

[0057] As discussed herein, minor variations in the amino acid sequences of antibody or immunoglobulin molecules are considered to be covered by the inventive concepts disclosed and claimed herein, provided that the variations in the amino acid sequences maintain at least 85% sequence similarity. Antibodies disclosed herein may be modified in a specific way to alter peptide characteristics without affecting their physiological activity. For example, alterations and / or deletions of certain amino acids may not affect the physiological activity of the antibodies disclosed herein (i.e., their efficacy in detecting PRRSV). Specifically, conserved substitutions of amino acids are expected. For example, it is reasonable to expect substitutions of leucine (L) for isoleucine (I) or valine (V), aspartic acid (D) for glutamic acid (E), threonine (T) for serine (S), or an amino acid for an amino acid of a related structure, which do not significantly affect the binding or properties of the amino acid molecule, particularly when the substitution does not involve the framework site of the amino acid. By analyzing the specific activities of peptide derivatives, it can be confirmed whether changes in amino acids affect a functional peptide. Those skilled in the art can prepare antibody fragments or analogs. Preferably, the amine or carboxyl terminus of the fragment or derivative is located near the functional domain.

[0058] Anti-PRRSV antibody 572 is indicated as antibody 572, no. 572 or 572; anti-PRRSV antibody 277 is indicated as antibody 277, no. 277 or 277.

[0059] (i) Antibody preparation

[0060] According to certain embodiments disclosed in this disclosure, the antibody is derived from fusion tumor strain 572 and named antibody 572. In these embodiments, the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, CDR-L3, CDR-VH (heavy chain variant domain) and CDR-VL (light chain variant domain) of antibody 572 include the amino acid sequences shown in the sequence listing.

[0061] The anti-PRRSV antibody of the present invention can be manufactured by the manufacturing method described below. That is, for example, a conjugate of PRRSV, a portion thereof, or a portion thereof, with a suitable carrier substance (e.g., bovine serum albumin, etc.) for enhancing the antigenicity of the antigen is used, along with an immunoactivator (Freund's complete or incomplete adjuvant, etc.) as needed, to immunize non-human mammals such as antibody-producing mice. PRRSV can be either natural PRRSV or recombinant PRRSV. Alternatively, immunosensitization can be achieved by introducing a gene encoding PRRSV and then administering it to animal cells that overexpress PRRSV on their cell surface. Monoclonal antibodies can be obtained by the following steps: culturing fusion tumors obtained by fusing antibody-producing cells obtained from self-immunized animals with myeloma lineage cells (myeloma cells) that lack the ability to produce self-antibodies, and selecting pure lines that can produce monoclonal antibodies that show specific affinity for the antigen used for immunization.

[0062] Alternatively, the antibody of the present invention (i.e., antibody 572) can be prepared by DNA selection. The DNA encoding the antibody of the present invention can be readily isolated and sequenced using conventional steps, such as using oligonucleotide probes capable of specifically binding to the genes encoding the heavy and light chains of the antibody of the present invention. The DNA sequences encoding antibody 572 used herein, namely CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3, are also shown in the sequence listing provided below. Once isolated, the DNA can be placed into an expression vector. It is then transfected into host cells and cultured under suitable conditions for expression. The term "expression vector" refers to a nucleic acid molecule capable of transporting another nucleic acid linked thereto. In one embodiment, the vector used herein is an expression vector and may be a "plast," which refers to a circular double-stranded DNA loop capable of linking additional DNA fragments. In another embodiment, the vector used herein is an expression vector and may be a viral vector, wherein additional DNA fragments may be linked to a viral genome to express the antibody. The vectors disclosed herein can self-replicate in the host cells they are introduced into (e.g., bacterial vectors with bacterial origins of replication and free mammalian vectors), or they can integrate into the host cell's genome after introduction, thereby replicating together with the host genome (e.g., non-attachment mammalian vectors). Host cells, such as E. coli cells, yeast cells, insect cells, HEK293 cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells, do not produce immunoglobulins, thereby enabling the synthesis of desired antibodies through recombinant host cells.

[0063] All degenerate nucleotide sequences are covered within the scope of this invention, provided that the peptide / polypeptide / protein of this invention encoded by the nucleotide sequence (e.g., the CDR, VH region, or VL region of this invention) retains the desired activity or function. The term "degenerate nucleotide sequence" refers to a nucleotide sequence comprising one or more degenerate codons (compared to a reference polynucleotide molecule encoding a polypeptide). Degenerate codons may contain nucleotides with different triplets but encode the same amino acid residue (i.e., GAU and GAC triplets each encoding aspartic acid (Asp)).

[0064] Depending on the application, the antibodies of the present invention or the DNA encoding the antibodies can be used to prepare chimeric antibodies (e.g., bispecific antibodies) and / or antibody fragments derived therefrom.

[0065] (ii) Method for detecting porcine reproductive and respiratory syndrome virus (PRRSV)

[0066] According to some embodiments, a method for detecting PRRSV includes the following steps: (a) preparing a sample; (b) contacting the sample with the antibody of the present invention or its antigen-binding fragment; and (c) determining the signal obtained after detection. The sample may first be lysed by methods familiar to those skilled in the art, such as freezing and thawing, ultrasonic treatment, pressure, enzymes, detergents, or combinations thereof. The virus in the sample is then detected by the present method or antibody using a suitable detection method, such as: Western ink dot assay, chemiluminescent microparticle immunoassay (CMIA), chemiluminescent immunoassay (CLIA), lateral fluid immunoassay (LFIA), or enzyme-conjugated immunosorbent assay (ELISA), or a biochip device (e.g., Bio-FET).

[0067] Several embodiments are presented below to illustrate certain aspects of this disclosure, enabling those skilled in the art to practice the invention. These embodiments should not be construed as limiting the scope of the invention. It is believed that those skilled in the art will be able to fully utilize and practice this disclosure without excessive interpretation after reading the description herein. All publicly available documents cited herein are incorporated herein by reference in their entirety.

[0068] Example

[0069] Example 1

[0070] Preparation of anti-PRRSV antibody

[0071] 1.1 Preparation of antigen

[0072] Viral lysates of PRRSV, porcine pseudorabies virus (denoted as S1462), porcine coronavirus (denoted as NTU), and porcine circovirus type 2 (denoted as 110-873S) were obtained from Dr. Ming-Tang Chiu of National Pingtung University of Science and Technology (NPUST). The unit concentration of the viral lysates was determined using the Bradford protein assay (manufacturer: BIO-RAD, USA), and diluted with phosphate-buffered saline (PBS) filtered through 0.22 microns. The viral lysates were then aliquoted and stored at -80°C for later use.

[0073] 1.2 Immunization

[0074] To generate anti-PRRSV antibodies, female BALB / c mice aged 6-8 weeks were used. 100 μg (concentration of 1 μg / μL) of PRRSV virus lysate was emulsified with an equal volume of complete Freund's Adjuvant (Sigma-Aldrich, USA) and then administered to the mice via intraperitoneal injection.

[0075] Boosting was performed on days 14, 28, and 42 with 100 μg of PRRSV viral lysate in incomplete Freund's adjuvant (Sigma-Aldrich, USA). Prior to sacrifice, two injections of 50 μg of PRRSV viral lysate emulsified with incomplete Freund's adjuvant were administered, three days apart, to induce an antibody response.

[0076] 1.3 Fusion tumor preparation and antibody purification

[0077] Immediately at the end of the immunization process, mouse spleens were collected and fused with myeloma cells for use in fusion tumor preparation and subsequent semi-solid selection (ClonaCell Hybridoma kit, STEMCELL Technologies, USA). All procedures were performed in accordance with the manufacturer's instructions. Fusion tumor strains were proliferated in 96-well microvolume discs (Product No. 3788, Corning, USA) until cell fusion was achieved, and the supernatant was collected for ELISA testing (PRRS X3 Ab test, IDEXX, USA; described below) to examine antibody responses to PRRSV. Antibodies 572 and 277, which have high binding affinity to PRRSV, were selected and purified by protein G agarose gel resin (Cytiva, USA). These purified monoclonal antibodies were dialyzed against PBS buffered saline to remove glycine and concentrated using an Amicon Ultra-15 centrifugal filter (10 kDa, Merck Millipore, USA). The monoclonal antibody was stored at -80 °C for subsequent experiments.

[0078] 1.4 Determining the binding affinity of anti-PRRSV antibody

[0079] ELISA was performed to determine the binding affinity of these anti-PRRSV antibodies to PRRSV. First, 100 ng of PRRSV viral lysate, obtained from Dr. Ming-Tang Chiu of National Pingtung University of Science and Technology (NPUST), was coated onto a 96-well microplate. Then, 0.5 μg of anti-PRRSV antibody 572 or 277 was added to each well as a primary antibody, and the mixture was reacted at room temperature (RT) for 1 hour. After 1 hour of reaction, unbound antibodies were removed by washing with a wash buffer (Tris-buffered saline (TBS) containing 0.05% Tween 20). Then, 4000-fold diluted anti-mouse IgG-HRP (Jackson ImmunoResearch Laboratory, Code: 515-005-071, USA) was added as a secondary antibody, and the mixture was reacted at room temperature for 2 hours. Following washing with a washing buffer solution (Tris-buffered saline (TBS) containing 0.05% Tween 20), the optical density (OD) value was evaluated by measuring the absorbance at 450 nm (EZ Read 400 Microplate Reader, Biochrom, USA). In the results (not shown), anti-PRRSV antibodies 572 and 277 exhibited high OD450 values, indicating that antibodies 572 and 277 have high binding affinity for PRRSV; therefore, these two antibodies were selected. Furthermore, anti-PRRSV antibody 277 will be used as a control example in subsequent embodiments.

[0080] 1.5 Characteristics of selected strains of two anti-PRRSV antibodies

[0081] According to certain embodiments of this disclosure, the gene sequence of the antibody variant region is resolved from the mRNA extract of anti-PRRSV antibody 572. The gene sequence is then analyzed using IgBlast [the IgBlast tool is provided by the National Institutes of Health (NIH) (nih.gov)]. After obtaining the CDR information, the polynucleotide sequence (represented as a DNA sequence) is translated into a polypeptide sequence (represented as an amino acid sequence) using ExPASy Translate (a tool for translating nucleotide sequences into protein sequences, provided by the Swiss Institute of Bioinformatics (SIB)). The polynucleotide sequence (represented as DNA sequence) of the VH domain of 572 (SEQ ID NO: 8) was translated into the polypeptide sequence (represented as amino acid sequence) of SEQ ID NO: 6, with three CDR sequences of SEQ ID NO: 1 (CDR-H1), SEQ ID NO: 2 (CDR-H2), and SEQ ID NO: 3 (CDR-H3). The polynucleotide sequence (represented as DNA sequence) of the VL domain of 572 (SEQ ID NO: 9) was translated into the polypeptide sequence (represented as amino acid sequence) of SEQ ID NO: 7, with three CDR sequences of SEQ ID NO: 4 (CDR-L1), SEQ ID NO: 5 (CDR-L3), and CDR-L2 containing the Lys-Ala-Ser sequence.

[0082] Example 2

[0083] Quantitative Limits of Anti-PRRSV Antibodies

[0084] In order to determine the minimum amount or lowest concentration of PRRSV that can be quantified with appropriate accuracy and precision by antibodies 572 and 277 against porcine reproductive and respiratory syndrome virus (PRRSV), the quantification limits are confirmed as follows.

[0085] The lowest concentration of PRRSV that could be distinguished by anti-PRRSV antibodies 572 and 277 was examined using an ELISA test. First, 0.78–100 ng of PRRSV viral lysate was coated onto a 96-well microplate. Then, 1 μg of the designated anti-PRRSV antibody was added as a primary antibody to each well and reacted at room temperature for 1 hour. After removing unbound antibodies with a wash buffer, 2500x diluted anti-mouse IgG-HRP was added as a secondary antibody to each well and reacted at room temperature for 2 hours. After washing with a wash buffer, TMB was added and reacted at room temperature for 30 minutes. The OD value at 450 nm was evaluated using an EZ Read 400 microplate analyzer.

[0086] The quantitative limit assay results of anti-PRRSV antibodies 572 and 277 are listed in Table 1. The OD 450 values ​​are shown in Table 1, and the background values ​​are approximately 0.15-0.2. As shown in Table 2, the concentration of 572 is 1 μg; the concentration of 277 is 1 μg. Comparing the quantitative limit assay results of 572 and 277, both 572 and 277 have the ability to detect 0.78-100 ng of PRRSV viral lysate. By ELISA assay, anti-PRRSV antibody 277 can recognize PRRSV at concentrations of 0.78-100 ng. Anti-PRRSV antibody 572 can recognize PRRSV at concentrations of 25.00-100 ng.

[0087] Table 1. Results of quantification limit of PRRSV for each selected strain of anti-PRRSV antibody determined by ELISA. PRRSV viral lysate (ng) No. 572 No. 277 1 μg 1 μg 100 2.603 1.267 0.411 0.097 0.073 0.067 0.061 0.061 1.816 1.683 1.558 1.538 1.555 1.453 1.528 1.457 50.00 25.00 12.50 6.25 3.13 1.56 0.78

[0088] Example 3

[0089] Cross-reactivity of anti-PRRSV antibodies

[0090] Experiment 1

[0091] To confirm the specificity of anti-PRRSV antibodies 572 and 277, the cross-reactivity of antibodies 572 and 277 against PRRSV viral lysates and other viral lysates was confirmed by ELISA. First, lysates of PRRSV viral lysates (denoted as PRRSV), pseudorabies virus (denoted as S1462), and porcine coronavirus (denoted as NTU) were coated onto 96-well microplates. Then, purified anti-PRRSV antibodies 572 and 277 (original concentration 0.8-1.0 mg / mL) diluted 1:4000 were added to each well as primary antibodies, and the reaction was carried out at 4°C for 2 hours.

[0092] After removing unbound antibodies, 1:5000 diluted peroxidase-labeled goat anti-mouse IgG (Peroxidase AffiniPure Goat Anti-Mouse IgG, light chain specific (Code: 115-035-174)) was added as a secondary antibody and reacted at 4°C for 2 hours. After washing with washing buffer, TMB was added and the reaction was repeated. OD450 values ​​were recorded using an EZ Read 400 microdisk analyzer. Each viral lysate was tested in two independent replicates, labeled Group 1 and Group 2. The OD450 values ​​from Experiment 1 are listed in Table 2 below.

[0093] Test 2

[0094] To confirm the cross-reactivity of antibodies 572 and 277 to PRRSV viral lysates and other viral lysates, an ELISA assay was performed. First, 96-well microplates were coated with PRRSV viral lysates (denoted as PRRSV), porcine circovirus type 2 lysates (denoted as 110-873S), and porcine coronavirus (denoted as NTU). Then, purified anti-PRRSV antibodies 572 and 277 (original concentration 0.8-1.0 mg / mL) diluted 1:4000 were added to each well as primary antibodies, and the mixture was reacted at 4°C for 2 hours.

[0095] After removing unbound antibodies, 1:2000 diluted peroxidase-labeled goat anti-mouse IgG (Peroxidase AffiniPure Goat Anti-Mouse IgG, light chain specific (Code: 115-035-174)) diluted in PBS was added as a secondary antibody, and the mixture was reacted at 4°C for 2 hours. After washing with washing buffer, TMB was added and the mixture was reacted again. The OD450 values ​​were recorded using an EZ Read 400 microdisk analyzer. The OD450 values ​​from Experiment 2 are listed in Table 3 below.

[0096] As shown in Tables 2 and 3, the OD450 values ​​of antibodies 572 and 277 against PRRSV virus are higher than those against other viruses, indicating that antibodies 572 and 277 both have the ability to distinguish PRRSV from other viruses.

[0097] Table 2. Results of ELISA assays to examine the cross-reactivity of antibodies 572 and 277 to PRRSV virus lysate, pseudorabies virus lysate (denoted as S1462), and porcine coronavirus (denoted as NTU). Viral lysates No. 572 No. 277 PBS 2' Ab was diluted with PBS (1:2000) 2' Ab was diluted with PBS (1:2000). 2' Ab was diluted with PBS (1:2000). Group 1 2 1 2 1 2 PRRSV 0.595 0.611 0.154 0.091 0.073 0.072 0.058 0.060 S1462 0.091 0.100 0.063 0.088 0.080 0.089 0.054 0.052 NTU 0.322 0.312 0.076 0.072 0.083 0.119 0.058 0.054

[0098] Table 3. Results of ELISA assays to examine the cross-reactivity of antibodies 572 and 277 to PRRSV viral lysate, porcine circovirus type 2 lysate (denoted as 110-873S), and porcine coronavirus (denoted as NTU). Viral lysates No. 572 No. 277 PBS 2' Ab was diluted with PBS (1:2000). 2' Ab was diluted with PBS (1:2000). 2' Ab was diluted with PBS (1:2000). PRRSV 1.484 0.303 0.118 0.121 NTU 0.782 0.185 0.140 0.145 110-873S 0.183 0.17 0.162 0.153

[0099] Example 4

[0100] Anti-PRRSV antibodies immobilized on a biosensor field-effect transistor (Bio-FET) were used to detect PRRSV.

[0101] A Bio-FET has a transistor region and a detection region, wherein the detection region further includes a detection surface. In embodiments of the invention, the detection surface can be functionalized with an antibody or antigen-binding fragment of porcine reproductive and respiratory syndrome virus (PRRSV) capable of binding to it. Specifically, in this embodiment, anti-PRRSV antibodies 572 and 277 can be applied to a Bio-FET to detect PRRSV in a sample. The following examples demonstrate the determination of the cross-reactivity (e.g., specificity) and sensitivity of anti-PRRSV antibodies 572 and 277 using a Bio-FET.

[0102] The steps for coating the Bio-FET with anti-PRRSV antibody 572 or 277 are as follows:

[0103] 1. Antibody fixation a. Prepare 50 μL of a 100 ng / mL solution of anti-PRRSV antibody 572 or 277 in 10 mM Bis-tris propane (BTP) buffer. b. Immerse the surface of the Bio-FET overnight in the 100 ng / mL solution of anti-PRRSV antibody 572 or 277 at 4°C and 90% relative humidity.

[0104] 2. BSA Blocking a. Rinse the Bio-FET three times with 10 mM BTP buffer. b. Immerse the Bio-FET in 1% bovine serum albumin (BSA) prepared with pH 7, 1xPBS buffer for 30 minutes at 37°C. c. Rinse the Bio-FET three times with 10 mM BTP buffer, then rinse twice with deionized (DI) water, and then air dry. The Bio-FET is then ready for use.

[0105] The steps for detecting anti-PRRSV antibodies 572 or 277 are as follows: a. First, load 100 μL of 10 mM, pH 7 BTP buffer into the polydimethylsiloxane (PDMS) wells on the Bio-FET using a pipette. Then, allow the system to stabilize for 10 minutes before measuring the drain current-gate voltage (ID-VG) reaction. Furthermore, the system is considered stable only after obtaining three consecutively overlapping ID-VG curves, with the last ID-VG curve serving as the baseline for the next step of biosensing. b. Remove the BTP buffer from the wells. c. Load 100 μL of sample into the wells and allow hybridization. More specifically, the sample is coronavirus, pseudorabies virus, or PRRSV. Furthermore, samples are started at the lowest concentration and gradually increased to higher concentrations. d. After 10 minutes, remove the sample and rinse the wells five times with 100 μL of wash buffer (10 mM BTP containing 0.05% Tween-20) using a pipette. e. Next, rinse the wells five times with 100 μL of 10 mM, pH 7 BTP buffer using a pipette to remove any nonspecific binding. f. After rinsing, fill the wells with 100 μL of fresh 10 mM, pH 7 BTP buffer. After the system has stabilized for 10 minutes, begin measuring the ID-VG reaction.

[0106] 4.1 Sensitivity of anti-PRRSV antibodies 572 and 277 immobilized on Bio-FET

[0107] PRRSV viral lysates (denoted as 763-P8) at different dilutions of 10⁻³, 10⁻⁶, 10⁻⁹, and 10⁻¹² were used as test samples to test the sensitivity of antibodies 572 and 277. Additionally, pseudorabies virus diluted to 10⁻⁶ (denoted as S1462-P7-PRV) was used as a negative control. A lower detectable concentration indicates higher antibody sensitivity. When the target biomolecule (i.e., PRRSV) binds to immobilized antibody 572 or 277, the electrical signal of the Bio-FET (represented as the threshold voltage difference in the examples) increases; therefore, generally, a higher electrical signal indicates more biomolecules are bound.

[0108] Figure 1A shows the results of the sensitivity of antibody 572 to PRRSV (denoted as 763-P8). The sample was PRRSV (denoted as 763-P8) with different dilution ratios, including 10⁻³, 10⁻⁶, 10⁻⁹, and 10⁻¹² dilutions. Additionally, a 10⁻⁶ dilution of pseudorabies virus (denoted as S1462-P7-PRV) was used as a negative control. As shown in Figure 1A, the electrical signals (threshold voltage differences) of antibody 572 against the 10⁻³, 10⁻⁶, 10⁻⁹, and 10⁻¹² dilutions of PRRSV (denoted as 763-P8) were significantly higher than those against the negative control. Furthermore, the electrical signal of antibody 572 against the 10⁻¹² dilution of PRRSV (763-P8) was 3 times higher than that against the 10⁻⁶ dilution of pseudorabies virus (denoted as S1462-P7-PRV) negative control.

[0109] Figure 1B shows the results of the sensitivity of anti-PRRSV antibody 277 to PRRSV (denoted as 763-P8). The sample was PRRSV (denoted as 763-P8) at different dilutions, including 10⁻³, 10⁻⁶, 10⁻⁹, and 10⁻¹² dilutions. Additionally, a 10⁻⁶ dilution of pseudorabies virus (denoted as S1462-P7-PRV) was used as a negative control. As shown in Figure 1B, only anti-PRRSV antibody 277 showed significantly higher electrical signals for PRRSV (denoted as 763-P8) at 10⁻³ and 10⁻⁶ dilutions than the negative control, while the electrical signals for PRRSV (denoted as 763-P8) at 10⁻⁹ and 10⁻¹² dilutions were even significantly lower than the negative control. Furthermore, when using only a 10⁻³ dilution of PRRSV, the electrical signal of anti-PRRSV antibody 277 against PRRSV (denoted as 763-P8) was 3 times higher than that of the negative control, i.e., a 10⁻⁶ dilution of porcine coronavirus (denoted as FCOV-P7). Therefore, the results indicate that antibody 572 on Bio-FET is more sensitive than antibody 277.

[0110] 4.2 Cross-reactivity of anti-PRRSV antibodies 572 and 277 immobilized on Bio-FET

[0111] To determine the specificity of antibody 572 immobilized on Bio-FET, Bio-FET was used to determine the cross-reactivity of antibody 572 immobilized on Bio-FET against PRRSV (denoted as 763-P8) and other viruses, including pseudorabies virus (denoted as S1462-P7-PRV), porcine coronavirus (denoted as FCOV-P7), and porcine circovirus type 2 (denoted as 110-873S). When the antibody showed a low cross-reactivity, it indicated that the antibody had high specificity.

[0112] Figure 2 shows the results of cross-reactivity between anti-PRRSV antibody 572 and PRRSV (denoted as 763-P8), pseudorabies virus (denoted as S1462-P7-PRV), porcine coronavirus (denoted as FCOV-P7), and porcine circovirus type 2 (denoted as 110-873S). These samples were used at dilutions of 10⁻³ and 10⁻⁶. As shown in Figure 2A, the electrical signal of anti-PRRSV antibody 572 against a 10⁻⁶ dilution of PRRSV (denoted as 763-P8) was significantly higher than that against a 10⁻⁶ dilution of pseudorabies virus (denoted as S1462-P7-PRV), a 10⁻⁶ dilution of porcine coronavirus (denoted as FCOV-P7), and a 10⁻⁶ dilution of porcine circovirus type 2 (denoted as 110-873S). Furthermore, the anti-PRRSV antibody 572 showed a 3-fold higher electrical signal against a 10⁻⁶ dilution of PRRSV (denoted as 763-P8) than against a 10⁻⁶ dilution of pseudorabies virus (denoted as S1462-P7-PRV).

[0113] As mentioned earlier, both antibodies 572 and 277 exhibited high specificity and sensitivity for PRRSV when applied to ELISA. However, when applied to Bio-FET, antibody 572 showed high sensitivity to PRRSV, while antibody 277 did not. This indicates that antibody 572 is not only applicable to ELISA but also a good candidate for detecting PRRSV in the Bio-FET field.

[0114] Application

[0115] For applications in detecting PRRSV, anti-PRRSV antibodies 572 and 277 can be used to detect animals (e.g., pigs) infected with PRRSV by using assays such as Western ink dot assay, CMIA, CLIA, LFIA, ELISA, and can be used to prepare compositions for detecting PRRSV in animals or samples from animals, such as for biochip devices (e.g., Bio-FET) or for detection kits for Western ink dot assay, CMIA, CLIA, LFIA, ELISA, or biochip devices. [Simplified Explanation of the Diagram]

[0044] This disclosure can be more fully understood after reading the following detailed description of the embodiments and referring to the drawings.

[0045] Figure 1A illustrates the sensitivity of anti-PRRSV antibody 572 (denoted as no. 572) to PRRSV (denoted as 763-P8) or pseudorabies virus (denoted as S1462-P7-PRV) when applied to Bio-FET.

[0046] Figure 1B illustrates the sensitivity of anti-PRRSV antibody 277 (denoted as no. 277) to PRRSV (denoted as 763-P8) or pseudorabies virus (denoted as S1462-P7-PRV) when applied to Bio-FET.

[0047] Figure 2 illustrates the cross-reactivity of anti-PRRSV antibody 572 (denoted as no. 572) with PRRSV (denoted as 763-P8), pseudorabies virus (denoted as S1462-P7-PRV), porcine coronavirus (denoted as FCOV-P7), or porcine circovirus type 2 (denoted as 110-873S) when applied to Bio-FET.

Claims

1. An antibody or antigen-binding fragment thereof against porcine reproductive and respiratory syndrome virus (PRRSV), wherein the antibody or antigen-binding fragment thereof comprises: A heavy chain variation domain includes: a first heavy chain complementarity determination region (CDR-H1) containing sequence number 1, a second heavy chain complementarity determination region (CDR-H2) containing sequence number 2, and a third heavy chain complementarity determination region (CDR-H3) containing sequence number 3; and a light chain variation domain includes: a first light chain complementarity determination region (CDR-L1) containing sequence number 4, a second light chain complementarity determination region (CDR-L2) containing the Lys-Ala-Ser sequence, and a third light chain complementarity determination region (CDR-L3) containing sequence number 5.

2. The antibody or antigen-binding fragment thereof as claimed in claim 1, wherein the heavy chain variant domain comprises the sequence numbered 6.

3. The antibody or antigen-binding fragment thereof as claimed in claim 1, wherein the light chain variant domain comprises the sequence numbered 7.

4. An isolated nucleic acid encoding an antibody or an antigen-binding fragment thereof as claimed in claim 1, wherein the isolated nucleic acid encoding the antibody or an antigen-binding fragment thereof comprises a first segment encoding the heavy chain variant domain and a second segment encoding the light chain variant domain.

5. The isolated nucleic acid as claimed in claim 4, wherein the first fragment encoding the heavy chain variant domain further comprises the sequence number 8.

6. The isolated nucleic acid as claimed in claim 4, wherein the second fragment encoding the light chain variant domain further comprises the sequence number 9.

7. A vector comprising the isolated nucleic acid as described in claim 4.

8. A host cell comprising the vector as described in claim 7.

9. A method for producing an antibody against porcine reproductive and respiratory syndrome virus (PRRSV) or an antigen-binding fragment thereof, comprising: (a) Culture the host cells as described in claim 8 under suitable conditions to express the antibody or its antigen-binding fragment; (b) recovering the antibody or its antigen-binding fragment, wherein the antibody or its antigen-binding fragment contains a heavy chain variant domain and a light chain variant domain.

10. The method of manufacturing an antibody against PRRSV or an antigen-binding fragment thereof as described in claim 9, wherein the heavy chain variant domain comprises the sequence numbered 6.

11. The method of manufacturing an antibody against PRRSV or an antigen-binding fragment thereof as described in claim 9, wherein the light chain variant domain comprises the sequence numbered 7.

12. A method for detecting porcine reproductive and respiratory syndrome virus (PRRSV), comprising: Contact a sample with the antibody or its antigen-binding fragment as described in claim 1.

13. The method as described in claim 12, wherein the heavy chain variant domain contains the sequence number 6.

14. A method as described in claim 12, wherein the light chain variant domain comprises the sequence number 7.

15. A bio-field effect transistor (Bio-FET) comprising: a transistor region; and a detection region, wherein the detection region includes a detection surface functionalized with an antibody or antigen-binding fragment thereof for binding porcine reproductive and respiratory syndrome virus (PRRSV), wherein the antibody or antigen-binding fragment thereof comprises: a heavy chain variation domain comprising: a first heavy chain complementarity-determining region (CDR-H1) containing sequence number 1, a second heavy chain complementarity-determining region (CDR-H2) containing sequence number 2, and a third heavy chain complementarity-determining region (CDR-H3) containing sequence number 3; and a light chain variation domain comprising: a first light chain complementarity-determining region (CDR-L1) containing sequence number 4, a second light chain complementarity-determining region (CDR-L2) containing a Lys-Ala-Ser sequence, and a third light chain complementarity-determining region (CDR-L3) containing sequence number 5.

16. The biofield-effect transistor as claimed in claim 15, wherein the heavy chain variant domain comprises the sequence number 6.

17. The biofield-effect transistor as claimed in claim 15, wherein the light chain variation domain comprises the sequence number 7.

18. A method for detecting porcine reproductive and respiratory syndrome virus (PRRSV) using a bio-field effect transistor (Bio-FET), the method comprising: (a) Binding a sample to an antibody or antigen-binding fragment thereof immobilized on a detection surface of the biofield-effect transistor, wherein the antibody or antigen-binding fragment comprises: a heavy chain variation domain comprising: a first heavy chain complementarity-determining region (CDR-H1) containing sequence number 1, a second heavy chain complementarity-determining region (CDR-H2) containing sequence number 2, and a third heavy chain complementarity-determining region (CDR-H3) containing sequence number 3; and a light chain variation domain comprising: a first light chain complementarity-determining region (CDR-L1) containing sequence number 4, a second light chain complementarity-determining region (CDR-L2) containing a Lys-Ala-Ser sequence, and a third light chain complementarity-determining region (CDR-L3) containing sequence number 5; and (b) Analyzing an electrical signal obtained from the biofield-effect transistor.

19. The method as described in claim 18, wherein the heavy chain variant domain contains the sequence number 6.

20. The method as described in claim 18, wherein the light chain variant domain contains the sequence number 7.

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