A monoclonal antibody against Avibacterium paragallinarum and its application

Abstract

The present invention discloses a monoclonal antibody against Avibacterium paragallinarum and its application. The monoclonal antibody comprises a light chain variable region and a heavy chain variable region, the amino acid sequence of HCDR1 of the monoclonal antibody is shown in SEQ ID NO:9, the amino acid sequence of HCDR2 is shown in SEQ ID NO:10, the amino acid sequence of HCDR3 is shown in SEQ ID NO:11, the amino acid sequence of LCDR1 is shown in SEQ ID NO:3, the amino acid sequence of LCDR2 is shown in SEQ ID NO:4, and the amino acid sequence of LCDR3 is shown in SEQ ID NO:5. The monoclonal antibody provided by the present invention can specifically target Avibacterium paragallinarum, has high affinity, and can be used for the enrichment, separation and / or detection of Avibacterium paragallinarum; the detection method constructed based on the monoclonal antibody is simple and fast to operate, has strong specificity, and is suitable for rapid detection of large batches of samples.

Description

Technical Field

[0001] The invention belongs to the technical field of pathogen separation and detection, and specifically relates to a monoclonal antibody against Avibacterium paragallinarum and application thereof. Background Art

[0002] Infectious coryza (IC) is an acute respiratory infectious disease of chickens caused by Avibacterium paragallinarum, which is associated with decreased egg production and slow growth in laying hens. The clinical symptoms of the disease generally include runny nose, facial swelling, tearing, conjunctivitis and other symptoms. The disease has a high mortality rate after mixed infection with other pathogens, and the disease develops and spreads rapidly, causing great economic losses to the poultry industry. The clinical symptoms of sick chickens are usually tearing, runny nose and swelling of the infraorbital sinus on one or both sides. However, because the symptoms of IC are similar to those of diseases such as chicken cholera and fowl pox, if the chickens are not vaccinated in advance, when sick chickens with similar symptoms to IC appear in the farm, it may not be possible to immediately identify the pathogen. Therefore, to confirm whether IC is infected, it is necessary to take samples of sick chickens, separate and identify the pathogens, and then determine the bacteria that infect the chickens.

[0003] Direct detection of A. paragallinarum in samples will be affected by a variety of opportunistic bacteria and other components. For the detection of A. paragallinarum in samples, it is necessary to first use sample pretreatment methods to remove interfering substances and background interference that is difficult to ignore, so as to improve the sensitivity and specificity of subsequent detection methods. In addition, before detection, the target bacteria in the sample need to be concentrated and enriched to reach the detection limit of the detection method. Traditional culture methods require 18-24 hours to cultivate the concentration of the target bacteria to the detection limit of the subsequent detection method. Immunological detection technology mainly relies on the specific recognition of antigens and antibodies, and detects unknown antigens or antibodies through known antibodies or antigens. Among them, the double antibody sandwich enzyme-linked immunosorbent assay (ELISA) is an immunoassay method based on immunoenzyme technology, by connecting known antibodies to solid phase carriers, the antigen to be tested is combined with it and then combined with detection antibodies and enzyme-labeled secondary antibodies. It can easily obtain results without special equipment and has the advantages of rapidity and strong specificity. It is suitable for large-scale rapid detection, and rapid detection products based on this technology are hot topics in clinical safety and rapid detection and analysis research. Summary of the invention

[0004] In order to solve the defects of the prior art in which the pretreatment steps are complicated and it is difficult to obtain high sensitivity and specificity when directly detecting Avibacterium paragallinarum in food, the present invention provides a monoclonal antibody against Avibacterium paragallinarum and application thereof.

[0005] The present invention solves the above technical problems through the following technical solutions.

[0006] The first aspect of the present invention provides a monoclonal antibody against Avibacterium paragallinarum, which comprises a light chain variable region and a heavy chain variable region, the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, the light chain variable region comprises LCDR1, LCDR2 and LCDR3, the amino acid sequence of the HCDR1 is shown in SEQ ID NO:9, the amino acid sequence of the HCDR2 is shown in SEQ ID NO:10, the amino acid sequence of the HCDR3 is shown in SEQ ID NO:11, the amino acid sequence of the LCDR1 is shown in SEQ ID NO:3, the amino acid sequence of the LCDR2 is shown in SEQ ID NO:4, and the amino acid sequence of the LCDR3 is shown in SEQ ID NO:5.

[0007] In one embodiment of the present invention, the framework region of the light chain variable region and / or the heavy chain variable region is a murine framework region.

[0008] In one embodiment of the invention, the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:6 or has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with SEQ ID NO:6.

[0009] In one embodiment of the invention, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:12 or has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with SEQ ID NO:12.

[0010] In one embodiment of the present invention, the monoclonal antibody is a full-length antibody, Fab, Fab', F(ab')2 or Fv, and the Fv is preferably scFv.

[0011] In one embodiment of the present invention, the monoclonal antibody is a full-length antibody.

[0012] In one embodiment of the present invention, the heavy chain constant region of the antibody is preferably derived from a murine heavy chain IgG2a constant region or a variant thereof, and the light chain constant region is preferably derived from a murine light chain κ chain constant region or a variant thereof.

[0013] In one embodiment of the present invention, the amino acid sequence of the light chain of the monoclonal antibody is as shown in SEQ ID NO:2; and / or, the amino acid sequence of the heavy chain of the monoclonal antibody is as shown in SEQ ID NO:8.

[0014] The second aspect of the present invention provides an isolated nucleic acid encoding the monoclonal antibody according to the first aspect of the present invention.

[0015] In one embodiment of the present invention, the nucleotide sequence encoding the light chain of the monoclonal antibody is shown as SEQ ID NO: 1; and / or, the nucleotide sequence encoding the heavy chain of the monoclonal antibody is shown as SEQ ID NO: 7.

[0016] The third aspect of the present invention provides a recombinant expression vector, which comprises the isolated nucleic acid as described in the second aspect of the present invention.

[0017] In one embodiment of the present invention, the recombinant expression vector is a plasmid, a cosmid, a phage or a viral vector, and the viral vector is preferably a retroviral vector, a lentiviral vector, an adenoviral vector or an adeno-associated viral vector.

[0018] The fourth aspect of the present invention provides a transformant, which comprises the isolated nucleic acid as described in the second aspect of the present invention or the recombinant expression vector as described in the third aspect of the present invention, and the host cell of the transformant is a prokaryotic cell or a eukaryotic cell.

[0019] In one embodiment of the present invention, the eukaryotic cell is a yeast cell or a mammalian cell.

[0020] In one embodiment of the present invention, the host cell is Escherichia coli, Pichia pastoris, CHO cells or HEK293 cells.

[0021] A fifth aspect of the present invention provides a method for preparing monoclonal antibodies against Avibacterium paragallinarum, the method comprising the following steps:

[0022] The transformant according to the fourth aspect of the present invention is cultured, and the monoclonal antibody is obtained from the culture.

[0023] The sixth aspect of the present invention provides the use of the monoclonal antibody described in the first aspect of the present invention, the isolated nucleic acid described in the second aspect of the present invention, the recombinant expression vector described in the third aspect of the present invention, and the transformant described in the fourth aspect of the present invention in the preparation of a kit for enriching, isolating and / or detecting Avibacterium paragallinarum.

[0024] In one embodiment of the present invention, the kit comprises the monoclonal antibody; and / or the A. paragallinarum is selected from one or more of serotype A A. paragallinarum, serotype B A. paragallinarum and serotype C A. paragallinarum.

[0025] In one embodiment of the present invention, the kit further comprises other reagents for enriching, separating and / or detecting Avibacterium paragallinarum based on ELISA or immunomagnetic bead technology.

[0026] In one embodiment of the present invention, the ELISA is a double antibody sandwich ELISA or an indirect ELISA, preferably a double antibody sandwich ELISA.

[0027] The seventh aspect of the present invention provides a kit, which comprises the monoclonal antibody as described in the first aspect of the present invention.

[0028] In one embodiment of the present invention, the kit further comprises other reagents for enriching, separating and / or detecting Avibacterium paragallinarum based on ELISA or immunomagnetic bead technology.

[0029] In one embodiment of the present invention, the ELISA is a double antibody sandwich ELISA or an indirect ELISA, preferably a double antibody sandwich ELISA.

[0030] The eighth aspect of the present invention provides a method for enriching, separating and / or detecting Avibacterium paragallinarum, the method comprising contacting a sample to be tested with the monoclonal antibody described in the first aspect of the present invention or the kit described in the seventh aspect of the present invention, and separating, enriching and / or detecting Avibacterium paragallinarum in the sample to be tested.

[0031] In one embodiment of the present invention, the method is an ELISA-based method; and / or, the method is for non-diagnostic purposes.

[0032] In one embodiment of the present invention, the ELISA is a double antibody sandwich ELISA or an indirect ELISA, preferably a double antibody sandwich ELISA.

[0033] In one embodiment of the present invention, the double antibody sandwich ELISA is selected from one or more of the following conditions:

[0034] (1) The capture antibody used in the double antibody sandwich ELISA is the monoclonal antibody, and / or the detection antibody used in the double antibody sandwich ELISA is an antibody obtained by immunizing a rabbit with one or more A. paragallinarum; the A. paragallinarum is preferably selected from one or more of serotype A A. paragallinarum, serotype B A. paragallinarum, and serotype C A. paragallinarum;

[0035] (2) The mass ratio of the capture antibody to the detection antibody is (2-5):1, preferably (2-3):1;

[0036] (3) The criterion for determining a positive result in the method is: 450 When the value was ≥ mean + 3 × standard deviation, it was judged as positive.

[0037] On the basis of being in accordance with the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0038] The reagents and raw materials used in the present invention are commercially available.

[0039] The positive and progressive effects of the present invention are:

[0040] The monoclonal antibody provided by the present invention can specifically target Avibacterium paragallinarum, has high affinity, and can be used for the enrichment, separation and / or detection of Avibacterium paragallinarum; the detection method constructed based on the monoclonal antibody is simple and fast to operate, has strong specificity, and is suitable for rapid detection of large batches of samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 Results of the detection ELISA with different working concentrations of capture and detection antibodies are shown.

[0042] Figure 2 The minimum detection limit of the A. paragallinarum double antibody sandwich ELISA detection method is shown. DETAILED DESCRIPTION

[0043] In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the molecular genetics, nucleic acid chemistry, chemistry, molecular biology, biochemistry, cell culture, microbiology, cell biology, genomics and recombinant DNA procedures used herein are conventional procedures widely used in the corresponding fields. At the same time, in order to better understand the present invention, the definitions and explanations of the relevant terms are provided below:

[0044] In the present invention, the letters in the amino acid sequence represent the single-letter abbreviations of amino acids known in the art, such as those described in J.Biol.Chem, 243, p3558 (1968): alanine: Ala-A, arginine: Arg-R, aspartic acid: Asp-D, cysteine: Cys-C, glutamine: Gln-Q, glutamic acid: Glu-E, histidine: His-H, glycine: Gly-G, asparagine: Asn-N, tyrosine: Tyr-Y, proline: Pro-P, serine: Ser-S, methionine: Met-M, lysine: Lys-K, valine: Val-V, isoleucine: Ile-I, phenylalanine: Phe-F, leucine: Leu-L, tryptophan: Trp-W, threonine: Thr-T.

[0045] In the present invention, the amino acid sequences of complementarity determining regions (CDRs) listed are shown according to the definition of IMGT numbering rules. However, it is well known to those skilled in the art that the CDR of an antibody can be defined by a variety of methods in the art, such as Chothia (Chothia et al. (1989) Nature 342:877-883, Al-Lazikani et al., "Standard conformations for the canonical structures of immunoglobulins", Journal of Molecular Biology, 273, 927-948 (1997)), based on the three-dimensional structure of the antibody and the topology of the CDR loop, Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, 4th Edition, US Department of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath), Contact (University College London), the international ImMunoGeneTics database (IMGT, World Wide Web imgt.cines.fr / ), and North based on affinity propagation clustering using a large number of crystal structures. CDR Definition. It will be appreciated by those skilled in the art that, unless otherwise specified, the terms "CDR" and "complementarity determining region" of a given antibody or region thereof (e.g., variable region) should be understood to encompass complementarity determining regions as defined by any of the above-mentioned known schemes described in the present invention.

[0046] Therefore, when referring to antibodies defined by specific CDR sequences defined in the present invention, the scope of the antibodies also covers antibodies whose variable region sequences contain the specific CDR sequences, but whose claimed CDR boundaries are different from the specific CDR boundaries defined in the present invention due to the application of different schemes (e.g., different assignment system rules or combinations). Although the scope of the present invention is based on the sequences shown in the definition according to the IMGT numbering rules, the amino acid sequences corresponding to the definition rules of other CDRs should also fall within the scope of the present invention.

[0047] In the present invention, the term "full-length antibody" is used interchangeably to refer to a glycoprotein comprising at least two heavy chains (HC) and two light chains (LC) interconnected by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated as VH in the present invention) and a heavy chain constant region. The heavy chain constant region consists of three domains CH1, CH2 and CH3. Each light chain consists of a light chain variable region (abbreviated as VL in the present invention) and a light chain constant region (abbreviated as CL in the present invention). The light chain constant region consists of one domain CL. Mammalian heavy chains are classified as α, δ, ε, γ and μ. Mammalian light chains are classified as λ or κ. Immunoglobulins containing α, δ, ε, γ and μ heavy chains are classified as immunoglobulins IgA, IgD, IgE, IgG and IgM. Complete antibodies form a "Y" shape. The stem of the Y is composed of the second and third constant regions of the two heavy chains (and the fourth constant region for IgE and IgM) combined together, and a disulfide bond (interchain) is formed in the hinge. Heavy chains γ, α and δ have a constant region consisting of three tandem (in a row) Ig domains, and a hinge region for increased flexibility; heavy chains μ and ε have a constant region consisting of four immunoglobulin domains. The second and third constant regions are called "CH2 domains" and "CH3 domains", respectively. Each arm of the Y includes the variable region of a single heavy chain bound to the variable and constant regions of a single light chain and the first constant region. The variable regions of the light and heavy chains are responsible for antigen binding.

[0048] In the present invention, "Fab fragment" consists of one light chain and CH1 and variable region of one heavy chain. The heavy chain of the Fab molecule cannot form a disulfide bond with another heavy chain molecule. The "Fc" region contains two heavy chain fragments including the CH2 and CH3 domains of the antibody. The two heavy chain fragments are held together by two or more disulfide bonds and by the hydrophobic interaction of the CH3 domain. "Fab' fragment" contains one light chain and a portion of one heavy chain including the VH domain and the CH1 domain and the region between the CH1 and CH2 domains, thereby forming an interchain disulfide bond between the two heavy chains of the two Fab' fragments to form a F(ab')2 molecule. "F(ab')2 fragment" contains two light chains and two heavy chains including a portion of the constant region between the CH1 and CH2 domains, thereby forming an interchain disulfide bond between the two heavy chains. Therefore, the F(ab')2 fragment consists of two Fab' fragments held together by a disulfide bond between the two heavy chains. The term "Fv" means an antibody fragment consisting of the VL and VH domains of a single arm of an antibody, but lacking a constant region.

[0049] In the present invention, the scFv refers to a single chain antibody fragment, which includes a heavy chain variable region, a light chain variable region and a connecting peptide of 15 to 20 amino acids. The VL and VH domains are paired to form a monovalent molecule by a connecting peptide that enables them to be produced as a single polypeptide chain [see, for example, Bird et al., Science 242: 423-426 (1988) and Huston et al., Proc. Natl. Acad. Sci. USA 85: 5879-5883 (1988)]. Such scFv molecules may have a general structure: NH2-VL-connecting peptide-VH-COOH or NH2-VH-connecting peptide-VL-COOH.

[0050] In the present invention, "nucleic acid" refers to a nucleotide chain of any length, and includes DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into the chain by DNA or RNA polymerase.

[0051] In the present invention, the term "recombinant expression vector" means a genetically modified oligonucleotide or polynucleotide construct that permits expression of mRNA, protein, polypeptide or peptide by a host cell when the construct comprises a nucleotide sequence encoding mRNA, protein, polypeptide or peptide, and the vector contacts the cell under conditions sufficient to allow mRNA, protein, polypeptide or peptide to be expressed in the cell. The vector of the present invention is generally not naturally occurring. However, portions of the vector may be naturally occurring. The recombinant expression vector of the present invention may comprise any type of nucleotide, including but not limited to DNA and RNA, which may be single-stranded or double-stranded, synthesized or partially obtained from a natural source, and which may contain natural, non-natural or altered nucleotides. The recombinant expression vector may comprise naturally occurring or non-naturally occurring nucleotides, or both types of connections. In exemplary aspects, altered nucleotides or non-naturally occurring nucleotides are connected without hindering transcription or replication of the vector.

[0052] The recombinant expression vector of the present invention can be any suitable recombinant expression vector, which can be used for transformation or transfection to deliver one or more genes or sequences of interest into any suitable host cell and preferably express the gene or sequence in the host cell. Suitable vectors include those designed for expansion and amplification or for expression or both, examples of vectors include but are not limited to viral vectors, naked DNA or RNA expression vectors, plasmids, cosmids or phage vectors, DNA or RNA expression vectors associated with cationic coagulants, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells, such as production cells.

[0053] In the present invention, the term "host cell" refers to any type of cell that can contain the nucleic acid or vector described herein. In exemplary aspects, the host cell is a eukaryotic cell, such as a plant, an animal, a fungus, or an alga; or it can be a prokaryotic cell, such as a bacterium or a protozoan.

[0054] In the present invention, the application scenarios of "non-diagnostic purposes" include but are not limited to: for example, as described in the present invention, the separation and enrichment of A. paragallinarum, the in vitro detection of the presence of A. paragallinarum in the laboratory; or as a positive antibody to screen other anti-A. paragallinarum antibodies.

[0055] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0056] The reagents, existing strains and their source information or preparation methods used in the following examples, and the information of the instruments are as follows Table 1-4:

[0057] Table 1. Source of strains

[0058]

[0059] Table 2 Preparation of main reagents and culture medium

[0060]

[0061]

[0062] Table 3 Main reagents and sources

[0063]

[0064]

[0065] Table 4 Main instruments and sources

[0066] Instrument Name source Emulsifier Purchased from Amalgamator Multifunctional ELISA reader Purchased from Biotek, USA Constant temperature culture shaker Purchased from Beijing Chuangxin Sicheng Co., Ltd. Constant temperature incubator Purchased from Thermo <![CDATA[CO2 Incubator]]> Purchased from ThermoScientific, USA Inverted microscope Purchased from Thermo Gel imaging system Purchased from Bio-Rad China Desktop low speed centrifuge Purchased from Hunan Xiangyi Laboratory Equipment Development Co., Ltd. Automatic autoclave Japan TOMY Company (X-500) Clean bench Zhejiang Sujing Purification Company (SW-CI-2FD) PCR instrument Eppendorf, Germany Constant temperature water bath Shanghai Jinghong Precision Instrument Constant temperature metal bath Shanghai Yiheng Scientific Instruments Drying box Shanghai Boxun Industrial Electronic Medical Equipment Factory Nucleic acid gel electrophoresis instrument Shanghai Xichuan Biotechnology Constant temperature incubator Purchased from Thermo Electronic balance Purchased from MettlerToledo, USA

[0067] Example 1 Preparation of Avibacterium paragallinarum Immunogen

[0068] (1) Preparation of Avibacterium paragallinarum immunogen:

[0069] a. Bacterial amplification: Inoculate 3 bacterial strains, CVCC 254, CVCC 256 and CVCC 257, into 10 mL TSB medium and amplify to OD 600 1, place in a 37°C, 5% CO2 incubator for 20-24 hours, and observe the growth characteristics.

[0070] b. Inactivation of bacterial solution: amplify 10mL of bacterial solution, and when OD 600 When the concentration of the bacterial strains was 1, the cells were inactivated with formaldehyde at a final concentration of 0.3% for more than 4 hours. 1 mL was taken and centrifuged at 12000 rpm for 5 minutes. The cells were washed with PBS for 5 times to remove the formaldehyde. The bacterial suspensions of the three strains were mixed in equal amounts and adjusted to 5.0×10 7 CFU / mL and place at 4℃ for later use.

[0071] c. Immunization of mice: Take equal amounts of bacterial solution and Freund's complete adjuvant and mix them evenly. Use an emulsifier to emulsify for 90 seconds. If an "oil-in-water" emulsion appears in the water, the emulsification is successful. The emulsified bacterial solution is placed at 4°C for use in immunizing mice.

[0072] (2) Preparation of polyclonal antibody against Avibacterium paragallinarum:

[0073] A. Bacterial amplification: Inoculate 3 bacterial strains, CVCC 254, CVCC 256 and CVCC 257, into 10 mL TSB medium and amplify to OD 600 1, place in a 37°C, 5% CO2 incubator for 20-24 hours, and observe the growth characteristics.

[0074] B. Inactivation of bacterial solution: amplify 10mL of bacterial solution, and when OD 600 When the concentration of the bacterial strains was 1, the cells were inactivated with formaldehyde at a final concentration of 0.3% for more than 4 hours. 1 mL was taken and centrifuged at 12000 rpm for 5 minutes. The cells were washed with PBS for 5 times to remove the formaldehyde. The bacterial suspensions of the three strains were mixed in equal amounts and adjusted to 5.0×10 7 CFU / mL and place at 4℃ for later use.

[0075] C. Immunization of rabbits: Take equal amounts of bacterial solution and Freund's complete adjuvant and mix them evenly. Use an emulsifier to emulsify for 90 seconds. If an "oil-in-water" emulsion appears in the water, the emulsification is successful. Place the emulsified bacterial solution at 4°C for use in immunizing rabbits.

[0076] Example 2 Animal Immunization Program

[0077] The primary immunization was bacterial solution and an equal amount of Freund's complete adjuvant, and the second to fourth immunizations were bacterial solution and an equal amount of Freund's incomplete adjuvant, and mice were immunized after emulsification for 120 seconds. The bacterial solution was diluted with physiological saline for booster intraperitoneal immunization. The immunization procedures for mice and rabbits are shown in Tables 5 and 6, respectively.

[0078] Table 5 Mouse immunization program

[0079]

[0080] Table 6 New Zealand White Rabbit Immunization Program

[0081] Immunization times Immunizing agent concentration (CFU / mL) Adjuvant Immune site Each immunization dose First immunization <![CDATA[1.0×10 9 ]]> Freund's complete adjuvant Subcutaneous 1mL Second immunization <![CDATA[1.0×10 9 ]]> Freund's incomplete adjuvant Subcutaneous 1mL The third immunization <![CDATA[1.0×10 9 ]]> Freund's incomplete adjuvant Subcutaneous 1mL Fourth immunization <![CDATA[1.0×10 8 ]]> Freund's incomplete adjuvant muscle 500μL

[0082] Example 3 Animal Serum Titer Determination

[0083] Balb / C mice were immunized, and mouse serum was collected after the fourth immunization at a ratio of 1:2×10 3 , 1:4×10 3 , 1:8×10 3 , 1:16×10 3 , 1:32×10 3 , 1:64×10 3 , 1:128×10 3 and 1:256×10 3 After proportional dilution, the serum titer was determined by indirect ELISA method, and the serum of healthy mice was used as negative control. The best immune mice were selected for cell fusion.

[0084] After booster immunization, mouse blood was collected by tail blood sampling, stored at 4°C overnight, centrifuged at 12,000 rpm for 5 min, upper serum was drawn, stored at -20°C for later use, and mouse serum titer was determined. The specific steps are as follows:

[0085] a. Inoculate the bacterial suspensions of CVCC 254, CVCC 256 and CVCC 257 into 10 mL TSB medium and amplify to OD 600 Perform formaldehyde inactivation for at least 4 h, transfer 100 μL of the culture medium to 1 mL of TSB liquid culture medium, and culture it in a shaking incubator at 37°C and 180 rpm for 18-24 h. Observe whether the bacterial solution is turbid.

[0086] b. Wash the inactivated 3 strains of Avibacterium paragallinarum 5 times with PBS. Dilute the bacterial solution 10-fold to 10 6 CFU / mL, add 100μL of diluent to each well of a 96-well plate, and coat the three bacterial strains CVCC 254, CVCC 256, and CVCC 257 separately at 4°C overnight. Wash 5 times with PBST, add gelatin and block at 37°C for at least 2h. Wash 5 times with PBST, dilute mouse serum with 5% FBS in PBST gradient (where the concentration of the monoclonal antibody is 2.51mg / mL), add 100μL of diluted serum to each well of a 96-well plate, and incubate at 37°C for 2h. Wash 5 times with PBST, dilute goat anti-mouse IgG (HRP labeled) at 1:20000, add 100μL to each well, and incubate at 37°C for 1h. Wash 5 times with PBST, add 100μL of TMB colorimetric solution to each well in the dark, color for 15min, add 50μL of stop solution, and read OD 450 , the mouse serum titer was analyzed and the results are shown in Table 7.

[0087] Table 7 OD of mouse serum after immunization450 Detection

[0088]

[0089] Three strains of CVCC 254, CVCC 256 and CVCC 257 were mixed in equal proportions and immunized into New Zealand white rabbits. After the fourth immunization, blood was collected from the ear vein and serum was separated (the concentration of the polyclonal antibody was 2.98 mg / mL). The serum was diluted by 10000, 20000, 40000, 80000, 160000, 320000, 640000, 1280000 and 2560000 times, and the titer was determined by indirect ELISA. Healthy rabbit serum was used as negative control. The rest of the steps were the same as above. The D 450 (P) and negative control D 450 The ratio of the value (N) (P / N) ≥ 2.1 was judged to be positive. The results are shown in Table 8 below.

[0090] Table 8 OD of rabbit serum after immunization 450 Detection

[0091]

[0092] Example 4 Preparation of Monoclonal Antibodies to Avibacterium paragallinarum

[0093] (1) Cultivation of myeloma cells (SP2 / 0)

[0094] a. Take out the frozen SP2 / 0 cells from the liquid nitrogen tank and put them into a 37℃ water bath to thaw the SP2 / 0 cells.

[0095] b. Operate in a clean bench, add the thawed SP2 / 0 cells to a centrifuge tube containing 8 mL of culture medium, centrifuge at 1000 rpm for 8 min, discard the supernatant, add 3 mL of DMEM complete culture medium, gently suspend the cells, inoculate into a cell bottle, add DMEM complete culture medium to 8 mL, and culture in a CO2 incubator. Subculture in time, and replace the culture medium the night before fusion.

[0096] (2) Preparation of feeder cells

[0097] a. Prepare feeder cells the night before the fusion of cells. Place the dissecting plate, scissors, forceps, 5mL syringe, needle, disposable gloves and other instruments in the ultra-clean bench for UV sterilization.

[0098] b. Kill Kunming mice by eyeballs, put them into a beaker containing 75% alcohol, immerse them completely in the alcohol, drain the alcohol off the surface of the mice, place them on a dissecting board, fix the limbs of the mice with needles, use tweezers to grab the skin on the lower side of the midline of the abdomen, cut a small hole horizontally with scissors, cut the skin upwards along the small hole, grab the skin on both sides with tweezers, separate them to both sides, fix the skin with a needle, and expose the peritoneum completely.

[0099] c. Pipette 5 mL of blank DMEM culture medium, gently lift the lower peritoneum with forceps, and quickly inject blank DMEM into the abdominal cavity from the lifted part.

[0100] d. Pat the abdomen about 50 times and draw ascites into a 15 mL centrifuge tube. Repeat this step three times. Centrifuge at 1000 rpm for 8 min, discard the supernatant, and blow off the cells with 35-40 mL of DMEM medium containing 20% ​​FBS HAT.

[0101] e. Resuspend the above feeder cells, pipette 200 μL into a 96-well cell plate, place in a 5% CO2 cell culture incubator, and observe the growth of the feeder cells every other day.

[0102] (3) Cell fusion

[0103] a. Preparation of myeloma cells: Prepare 4 bottles of SP2 / 0 cells, wash them with 3 mL of blank DMEM, discard the supernatant, gently blow down the SP2 / 0 cells with 5 mL of blank DMEM (to prevent cell breakage), centrifuge at 1000 rpm for 8 min, discard the supernatant, resuspend the SP2 / 0 cells with blank DMEM, and transfer them to a 50 mL centrifuge tube.

[0104] b. Preparation of spleen cells: Remove the eyeballs of mice and kill them. Place them in a beaker containing 75% alcohol. Pour the alcohol off the surface of the mice and place them on a dissecting board. Fix the limbs of the mice with needles and expose the abdomen. Use scissors and forceps to open the abdominal cavity and take out the spleen. The spleen of the immunized mice is more than 2 times larger than that of normal mice, and is red and black in shape. Place it in a 6-well plate and wash it with 5 mL of blank DMEM for later use.

[0105] c. Extract mouse spleen cells: Add 5 mL of blank DMEM to a 6-well plate. Gently clamp one end of the spleen with tweezers, take 5 mL of blank DMEM, pierce one end of the spleen, and gently clamp the spleen while injecting 5 mL of blank DMEM. The culture medium flushed out of the spleen is suspected to be pink. Repeat the above steps. When the spleen turns pink, discard the spleen. Transfer the spleen cell suspension to a 15 mL centrifuge tube, centrifuge at 1000 rpm for 8 minutes, discard the supernatant, add an appropriate amount of blank DMEM to resuspend the cells, and add to the 50 mL centrifuge tube containing SP2 / 0 cells.

[0106] d. Mix the spleen cells and SP2 / 0 cells, add 30 mL of preheated blank DMEM, centrifuge at 1000 rpm for 10 min, and discard the supernatant.

[0107] e. After centrifugation, the cells are divided into two layers, spleen cells are red, and SP2 / 0 cells are white. Gently tap the bottom of the fusion tube with the palm of your hand until the two types of cells are evenly mixed and appear pink.

[0108] f. Take out 1 mL of preheated fusion agent PEG and drip it into the fusion tube at a uniform speed within 45 seconds. Add PEG and let it act at room temperature for 90 seconds.

[0109] g. Add 15 mL of preheated blank DMEM to a 50 mL centrifuge tube to terminate the PEG reaction. The order of addition is: add 1 mL of blank DMEM within the first 30 s; then add 2 mL of blank DMEM within 30 s; and finally add 10 mL of blank DMEM within 120 s.

[0110] h. Add 15 mL of preheated blank DMEM, let stand for 10 min, centrifuge at 1000 rpm for 8 min, and discard the supernatant.

[0111] i. Add 10 mL of HAT medium containing 20% ​​FBS, gently resuspend the cells, then take 0.5 drops, 1 drop, 1.5 drops, 2 drops, 3 drops and 4 drops of the resuspended cells, resuspend them with 10 mL of HAT complete medium respectively, and spread them on the feeder cells prepared in advance, add 200 μL to each well, culture in a 5% CO2 incubator, and observe whether there are fused cells after 3-4 days.

[0112] (4) Screening of positive clones after fusion

[0113] a. About 3-4 days after fusion, observe all 96-well plates under a microscope and mark the number of single clone wells. After 2 weeks, aspirate 100 μL of cell supernatant and move it to the laboratory to determine the titer by ELISA method and replenish HAT medium.

[0114] b. ELISA steps are the same as the mouse serum titer determination, CVCC 254, CVCC 256 and CVCC 257 are mixed and coated respectively. The primary antibody is 90 μL of PBST containing 5% FBS and 10 μL of cell supernatant, and the other steps are the same as Example 3.

[0115] c. Determine cell specificity: Take 100 μL of cell supernatant, mark it clearly, transfer it to the laboratory, and use the indirect ELISA method to determine the cell specificity. Inoculate 7 common environmental pathogens such as Bacillus subtilis, Streptococcus uberis, Clostridium perfringens, Escherichia coli O157, Staphylococcus aureus, Proteus, and Salmonella pullorum into TSB medium, and culture at 37°C, 180 rpm constant temperature shaker until the bacterial solution concentration is OD 600 When the concentration is 1.0, add formaldehyde, inactivate at least 4 hours at 37°C, 180rpm constant temperature shaker, and wash 5 times with PBS. Resuspend the cells in the coating solution as the coating source. Take 10 μL of cell supernatant and 90 μL of PBST, mix them, dilute them in multiple ratios, and use them as the primary antibody. The rest of the steps are the same as in Example 3.

[0116] (5) Subcloning of positive hybridoma cells

[0117] a. Pick monoclonal cell lines with high titer and good growth status in cell supernatant for subcloning. First, mark the cell position of the positive clone under a microscope, use a 200μL pipette to transfer a small amount of cells to a 96-well cell plate, and perform gradient dilution with an incomplete culture medium containing 20% ​​FBS. Observe under an inverted microscope, select wells containing 90-120 cells per well, transfer the liquid in the well to 10mL of 20% FBS incomplete culture medium, and mix by pipetting. Then add dropwise to a 96-well cell plate containing feeder cells (100μ / well), and place at 37°C and 5%

[0118] Culture in a CO2 incubator. Observe the cell growth status in time and add the appropriate amount of culture medium within 3-5 days. When the cells grow to the size of a fingernail, perform screening and subcloning again until the positive rate of the cell supernatant in the monoclonal well reaches 100%. Expand the culture, freeze and mark at the same time.

[0119] b. On the 3rd to 4th day after cell fusion, all cell culture wells were examined under a microscope, and 96 wells with 1 to 2 monoclonal cells were marked. On the 8th to 9th day after cell fusion, the culture supernatant with monoclonal cells was selected for titer detection. Subsequently, monoclonal cell lines with high titer and good specificity were selected for subcloning.

[0120] (6) Preparation of mouse ascites

[0121] 0.5 mL of sterilized paraffin oil was injected into the peritoneal cavity of female Balb / C mice every 7 days for a total of 3 injections. Then 0.5 mL of subcloned positive cells and an equal volume of sterilized paraffin oil were injected into the peritoneal cavity of the mice. 7-15 days after injection, the physiological and mental state of the mice was observed several times a day. For mice with mental depression, abdominal distension, and fluctuation when pressed, ascites was collected with a sterile syringe needle when they were on the verge of death. After the ascites was allowed to stand for 1-2 hours, it was centrifuged at 4°C and 5000rpm for 30 minutes. After centrifugation, the ascites was divided into three layers, of which the middle layer was the yellow transparent ascites. In the clean bench, the ascites was collected with a pipette, stored in clean EP tubes, and frozen at -80°C.

[0122] Example 5 Purification of polyclonal antibodies

[0123] 1) Add 3 mL of New Zealand white rabbit serum and PBS to a 20 mL centrifuge tube, respectively, and vortex to mix. Place the centrifuge tube in an ice water bath, slowly add 6 mL of pre-cooled 50% saturated ammonium sulfate solution to the test tube, and let stand at 4°C for 12 h.

[0124] 2) After standing for 12 h, carefully remove the supernatant and transfer the precipitate to a 10 mL centrifuge tube. Centrifuge at 3000 r / min for 20 min at 4 °C. Collect the precipitate and dissolve it again in 3 mL PBS (same as step 1). Add 2 mL 40% saturated ammonium sulfate and stand at 4 °C for 1 h.

[0125] 3) Centrifuge the precipitate again (same as step 2), dissolve the precipitate in 3 mL PBS (same as step 1), and add 2 mL

[0126] 33% saturated ammonium sulfate, let stand at 4°C for 1 hour.

[0127] 4) Dissolve the precipitate with a small amount of PBS, mix thoroughly and place in a dialysis bag. Dialyze with PBS at 4°C for 72 hours, replacing PBS every 12 hours.

[0128] 5) Use Nessler's reagent to test the serum. When no ammonium ions are detected, it indicates that the purification is complete. Collect the purified antibodies.

[0129] The antibody concentration was determined to be 2.15 mg / mL and stored at -80°C until use.

[0130] Example 6 Monoclonal Antibody Analysis

[0131] 1. Determination of potency and specificity

[0132] The titer of the A. paragallinarum-specific monoclonal antibody was measured by ELISA. The inactivated bacterial solutions of three species of A. paragallinarum CVCC 254, CVCC 256 and CVCC 257 were mixed and coated using a coating buffer. The bacterial solutions were mixed and 100 μL per well was coated on a 96-well ELISA plate. The antibody detection steps were the same as those in Example 3. The collected ascites monoclonal antibody (1.38 mg / mL) was diluted in a gradient manner according to 1:10,000, 1:20,000, 1:40,000, 1:80,000, 1:160,000, 1:320,000, 1:640,000, 1:1,280,000, 1:2,560,000, 1:5,120,000, 1:10,240,000, 1:20,480,000, and 1:40,960,000. 100 μL of each concentration was added to each well as the primary antibody. The rest of the ELISA operation steps were the same as the mouse serum titer determination. At the same time, a positive and negative control was set up. The OD value was determined by the microplate reader. 450 The titer was determined by a P / N value ≥ 2.1, and the maximum dilution was the titer of the antibody. The standard laboratory strains of Bacillus subtilis, Streptococcus uberis, Clostridium perfringens, Escherichia coli O157, Staphylococcus aureus, Proteus and Salmonella pullorum were inoculated into TSB medium and cultured at 37°C and 180 rpm in a constant temperature shaker until the bacterial solution concentration was OD 600 When the concentration is 1.0, formaldehyde is added, and the cells are inactivated at 37°C, 180 rpm constant temperature shaker for at least 4 hours, and washed with PBS 5 times. The cells are resuspended in the coating solution as the coating source. The primary antibody is 10 μL mouse ascites + 90 μL 5% serum PBST. The secondary antibody concentration is 1:10000, and the remaining steps are the same as in Example 3.

[0133] After 4 subclonings, a monoclonal antibody was screened out and named 3-D8. The titer of ascites was determined by indirect ELISA method using Avibacterium paragallinarum CVCC 254, CVCC 256 and CVCC 257 as coating sources. The specific method was the same as above. From the results (Table 8), it can be seen that the titer of monoclonal antibody 3-D8 is relatively high. When diluted 5,120,000 times, the OD 450 It is still above 0.6, and the P / N value is ≥2.1. Therefore, the titer of 3-D8 is 5,120,000. Using inactivated Bacillus subtilis, Streptococcus uberis, Clostridium perfringens, Escherichia coli O157, Staphylococcus aureus, Proteus and Salmonella pullorum as coating sources, 3-D8 was diluted 50,000 times to measure the specificity of the antibody. The results (Table 9) showed that none of the antibodies reacted with the above 7 common foodborne pathogens, indicating that the antibody specificity was good.

[0134] Table 8 OD of monoclonal antibody 3-D8 450 Determination

[0135]

[0136]

[0137] Table 9 Specificity determination of monoclonal antibody 3-D8

[0138] Strain name <![CDATA[OD 450 ]]> Bacillus subtilis 0.046 Proteus vulgaris 0.069 Escherichia coli O157 0.045 Salmonella pullorum 0.054 Staphylococcus aureus 0.059 Streptococcus uberis 0.032 Clostridium perfringens 0.043 Negative 0.050

[0139] 2. Identification of Monoclonal Antibodies

[0140] The inactivated Avibacterium paragallinarum was used to coat a 96-well ELISA plate, and the monoclonal antibody subtype identification kit was used to identify the antibody subtype of each positive antibody collected. The primary antibody was diluted at a multiple of 1:10,000, and the secondary antibody was added at a dilution multiple of 1:300 using the antibody in the kit.

[0141] The results of subtype identification of monoclonal antibodies using an antibody subtype identification kit showed that the light chain type of the antibodies was all kappa type and the heavy chain type was IgG1 type.

[0142] Example 7 Determination and analysis of anti-Avibacterium paragallinarum monoclonal antibody sequences

[0143] The monoclonal antibody 3-D8 has the best titer and specificity. In order to identify the 3-D8 antibody sequence, Trizol reagent was used to isolate total RNA from hybridoma cells secreting monoclonal antibody 3-D8. Specific steps: 1) Trizol lysis: resuspend all bacteria with 1 mL Trizol, mix well by pipetting, and let stand for 3-5 minutes. (2) Chloroform phase separation: add 0.2 mL of chloroform (trichloromethane) to each sample, shake vigorously for 15 seconds, let it stand at room temperature for 3 minutes, and then centrifuge at 12,000 rpm for 15 minutes. (3) RNA precipitation: transfer the upper aqueous phase to a new 1.5 mL RNase-free EP tube, add 500 μL of isopropanol to precipitate the RNA molecules, and mix thoroughly by inverting. (4) RNA washing: gently aspirate the supernatant (be careful not to aspirate the white precipitate), drain on paper, add 1 mL of 75% ethanol (750 μL of anhydrous ethanol, 250 μL of DEPC water, prepared in advance) to the precipitate, and centrifuge at 7,500 rpm for 5 minutes. (5) RNA dissolution: discard the supernatant and dry for 10 min. Add appropriate amount of DEPC water to dissolve, place on ice, and measure OD 260 / OD 280 .

[0144] Then, the antibody constant region specific primers were used and reverse transcribed into cDNA using a Thermo reverse transcription kit, and PCR amplification was performed using the corresponding cDNA as a template using Ex Taq enzyme (Takara) and degenerate primers, followed by sequencing to obtain the variable region sequences of the heavy and light chains of the monoclonal antibody. The positions of the complementary determining regions (CDRs) were determined using the IMGT definition system.

[0145] In order to determine the antibody sequence, RNA was extracted from hybridoma cells, and then the variable region sequence was amplified by the degenerate primer method. The sequencing results showed that the sequence of the monoclonal antibody variable region was a gene fragment of the germline gene family (Table 10).

[0146] Table 10 Germline genes of variable regions of anti-Avibacterium paragallinarum monoclonal antibodies (IgBlast analysis)

[0147] MAb <![CDATA[V H ]]> <![CDATA[D H ]]> <![CDATA[J H ]]> <![CDATA[V K ]]> <![CDATA[J K ]]> 3-D8 12-1*01 2-14*01 4*01 6-23*01 5*01

[0148] Note: V H , D H , J H They are the V, D, and J germline gene segments of the heavy chain variable region. K , J K They are the V and J germline gene segments of the light chain variable region, respectively.

[0149] The sequencing results are as follows:

[0150] Light chain nucleotide sequence:

[0151]

[0152]

[0153] Note: The bold part is the variable region nucleotide sequence, and the non-bold part is the constant region nucleotide sequence.

[0154] Light chain amino acid sequence:

[0155]

[0156] Note: The bold part is the variable region amino acid sequence, and the non-bold part is the constant region amino acid sequence. The bold and underlined parts are the complementarity determining regions (CDRs).

[0157] in:

[0158] LCDR1: QDVGTA (SEQ ID NO: 3)

[0159] LCDR2: WAS (SEQ ID NO: 4)

[0160] LCDR3:QQYSSYPT (SEQ ID NO:5)

[0161] Light chain variable region amino acid sequence:

[0162] DIVMTQSHKFMSTSVGDRVSITCKASQDVGTAVAWYQQKPGQSPKLMIYWAST RHTGVPDRFTGSGSGTDFTLTISNVQYEDLADYFCQQYSSYPTFGAGTKLELK(SEQ ID NO:6)

[0163] Heavy chain nucleotide sequence:

[0164]

[0165]

[0166] Note: The bold part is the variable region nucleotide sequence, and the non-bold part is the constant region nucleotide sequence.

[0167] Heavy chain amino acid sequence:

[0168]

[0169] Note: The bold part is the variable region amino acid sequence, and the non-bold part is the constant region amino acid sequence. The bold and underlined parts are the complementarity determining regions (CDRs).

[0170] in:

[0171] HCDR1: GFLFSSYD (SEQ ID NO: 9)

[0172] HCDR2:ISGSGSKT(SEQ ID NO:10)

[0173] HCDR3:TRHSYRYVMDY(SEQ ID NO:11)

[0174] Heavy chain variable region amino acid sequence:

[0175] EVQLVESGGGLVKPGGSLKLSCAASGFLFSSYDMSWVRQTPEKRLEWVAKISGSG SKTHYPDTVKGRFTISRDNAKNTLFLQLSSLQSEDTAMYYCTRHSYRYVMDYWGQGT SVTVSS(SEQ ID NO:12)

[0176] Example 8 Establishment of anti-Avibacterium paragallinarum double antibody sandwich ELISA method

[0177] 1. Optimization of conditions for double antibody sandwich ELISA method

[0178] 100 μL of mouse ascites containing anti-Avibacterium paragallinarum monoclonal antibody (1.38 mg / mL) was added to a 96-well ELISA plate at a dilution ratio of 1:500, 1:1000, 1:2000, and 1:4000, and incubated at 37°C for 4 h and washed three times with 1×PBST; 200 μL of 10 g / μL gelatin was added to each well, blocked at 37°C for 2 h, and washed three times with PBST; then 100 μL of 10 7 CFU / mL of three strains (CVCC 254, CVCC 256 and CVCC 257 mixed in equal proportions) of A. paragallinarum bacterial solution, a negative control was set at the same time, incubated at 37°C for 1h, washed 3 times with PBST, and then diluted at a ratio of 1:2000, 1:4000, 1:8000, and 1:16000, and 100 μL of rabbit anti-A. paragallinarum polyclonal antibody storage solution (Example 5) containing 5% fetal bovine serum was added vertically, incubated at 37°C for 1h, and washed 3 times with PBST; then 100 μL of 1:2000 diluted HRP-labeled goat anti-rabbit secondary antibody was added to each well, incubated at 37°C for 45min, and washed 3 times with 1×PBST; finally, 100 μL of TMB substrate was added to each well, reacted at 37°C for 15min, and D was read using an enzyme reader. 450 Value, P / N ≥ 2.1 can be judged as positive.

[0179] Results: Figure 1 As shown, when the prepared anti-Avibacterium paragallinarum monoclonal antibody was diluted to 1:1000 and the anti-Avibacterium paragallinarum rabbit polyclonal antibody was diluted to 1:4000, the P / N value was the highest, so the above antibody dilutions were selected to establish the ELISA detection method.

[0180] 2. Determination of positive judgment criteria

[0181] Eight negative sera collected from blank BALB / C (SPF grade) mice (purchased from Shanghai Sibeifu Experimental Animal Co., Ltd.) were selected and the OD 450 The mean and standard deviation (s). 450 When the value is greater than the average value + 3s, it is judged as positive; otherwise, when the sample OD 450 When the value is less than the average value + 3s, it is judged as negative.

[0182] Results: The D of 8 negative samples was determined by the established double antibody sandwich ELISA method. 450 Value, calculated OD 450 The average value is 0.132, and the standard deviation (s) is 0.012, so the positive and negative critical value (average value + 3s) is determined to be 0.171 (Table 12). 450When the value is greater than 0.170 and P / N ≥ 2.1, it is judged as positive; otherwise, it is judged as negative.

[0183] Table 12 Mouse negative serum OD 450 Detection

[0184] Mouse negative serum number <![CDATA[OD 450 ]]> <![CDATA[OD 450 ]]> 1 0.129 0.118 2 0.125 0.153 3 0.136 0.129 4 0.13 0.148 5 0.141 0.144 6 0.127 0.132 7 0.125 0.127 8 0.131 0.121

[0185] 3. Sensitivity determination of double antibody sandwich ELISA

[0186] Avibacterium paragallinarum CVCC 254, CVCC 256 and CVCC 257 were mixed as the detection antigen and the concentration of the bacterial solution was adjusted to 10 7 , 10 6 , 10 5 , 10 4 , 10 3 , 10 2 , 10 1 CFU / mL. Optimize according to the above steps. When P / N ≥ 2.1, it can be considered as the minimum detection limit of this method, and the serum of healthy rabbits is used as a negative control.

[0187] Results: Figure 2 As shown, when the bacterial concentration is 1×10 5 CFU / mL, the mean P / N value was 2.31 (greater than 2.1), indicating that the minimum detection limit of this method was 1×10 5 CFU / mL.

[0188] 4. Specificity of double antibody sandwich ELISA

[0189] Bacillus subtilis, Streptococcus uberis, Clostridium perfringens, Escherichia coli O157, Staphylococcus aureus, Proteus and Salmonella pullorum were enriched for 10 7 CFU / mL. Optimize its specificity according to the above steps.

[0190] Results: When the double antibody sandwich ELISA method was used for detection, only the result was positive when Avibacterium paragallinarum was detected, while there was no cross reaction when detecting other 7 common foodborne bacteria including Escherichia coli and Salmonella, and the results were all negative (see Table 13), indicating that this method has good specificity.

[0191] Table 13 Specificity of double antibody sandwich ELISA Specificity detection

[0192]

[0193]

[0194] Although the specific embodiments of the present invention are described above, it should be understood by those skilled in the art that these are only examples, and various changes or modifications may be made to these embodiments without departing from the principles and essence of the present invention. Therefore, the protection scope of the present invention is limited by the appended claims.

Claims

1. A monoclonal antibody against Avibacterium paragallinarum, comprising a light chain variable region and a heavy chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3, characterized in that: The amino acid sequence of the HCDR1 is shown in SEQ ID NO: 9, the amino acid sequence of the HCDR2 is shown in SEQ ID NO: 10, the amino acid sequence of the HCDR3 is shown in SEQ ID NO: 11, the amino acid sequence of the LCDR1 is shown in SEQ ID NO: 3, the amino acid sequence of the LCDR2 is shown in SEQ ID NO: 4, and the amino acid sequence of the LCDR3 is shown in SEQ ID NO: 5; Wherein, the amino acid sequence of the LCDR2 is WAS.

2. The monoclonal antibody according to claim 1, characterized in that The framework region of the light chain variable region and / or the heavy chain variable region is a murine framework region.

3. The monoclonal antibody according to claim 2, characterized in that The amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 6 or has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with SEQ ID NO: 6; And / or, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 12 or has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with SEQ ID NO:

12.

4. The monoclonal antibody according to any one of claims 1 to 3, characterized in that The monoclonal antibody is a full-length antibody, Fab, Fab', F(ab')2 or Fv.

5. The monoclonal antibody according to claim 4, characterized in that The Fv is a scFv.

6. The monoclonal antibody according to claim 4, characterized in that The monoclonal antibody is a full-length antibody.

7. The monoclonal antibody according to claim 6, characterized in that The heavy chain constant region of the monoclonal antibody is derived from the murine heavy chain IgG2a constant region, and the light chain constant region is derived from the murine light chain κ chain constant region.

8. The monoclonal antibody according to claim 7, characterized in that The amino acid sequence of the light chain of the monoclonal antibody is shown in SEQ ID NO: 2; and / or, the amino acid sequence of the heavy chain of the monoclonal antibody is shown in SEQ ID NO:

8.

9. An isolated nucleic acid, characterized in that The isolated nucleic acid encodes the monoclonal antibody according to any one of claims 1 to 8.

10. The isolated nucleic acid according to claim 9, wherein The nucleotide sequence encoding the light chain of the monoclonal antibody is shown in SEQ ID NO: 1; and / or, the nucleotide sequence encoding the heavy chain of the monoclonal antibody is shown in SEQ ID NO:

7.

11. A recombinant expression vector, characterized in that: The recombinant expression vector comprises the isolated nucleic acid according to claim 9 or 10.

12. The recombinant expression vector according to claim 11, characterized in that The recombinant expression vector is a plasmid, a cosmid or a viral vector.

13. The recombinant expression vector according to claim 12, characterized in that: The viral vector is a retroviral vector, an adenoviral vector or an adeno-associated viral vector.

14. The recombinant expression vector according to claim 12, characterized in that: The viral vector is a lentiviral vector.

15. The recombinant expression vector according to claim 11, characterized in that: The recombinant expression vector is a bacteriophage.

16. A transformant, characterized in that: The transformant comprises the isolated nucleic acid according to claim 9 or 10 or the recombinant expression vector according to any one of claims 11 to 15, and the host cell of the transformant is a prokaryotic cell or a eukaryotic cell.

17. The transformant according to claim 16, wherein The eukaryotic cell is a yeast cell or a mammalian cell.

18. The transformant according to claim 16 or 17, characterized in that The host cell is Escherichia coli, Pichia pastoris, CHO cell or HEK293 cell.

19. A method for preparing monoclonal antibodies against Avibacterium paragallinarum, characterized in that: The method comprises the following steps: Cultivating the transformant according to any one of claims 16 to 18, and obtaining the monoclonal antibody from the culture.

20. Use of the monoclonal antibody according to any one of claims 1 to 8, the isolated nucleic acid according to claim 9 or 10, the recombinant expression vector according to any one of claims 11 to 15, or the transformant according to any one of claims 16 to 18 in the preparation of a kit for enriching, isolating and / or detecting Avibacterium paragallinarum.

21. The use according to claim 20, characterized in that The kit comprises the monoclonal antibody; and / or the A. paragallinarum is selected from one or more of serotype A A. paragallinarum, serotype B A. paragallinarum and serotype C A. paragallinarum.

22. The use according to claim 21, characterized in that The kit also includes other reagents for enriching, isolating and / or detecting Avibacterium paragallinarum based on ELISA or immunomagnetic bead technology.

23. The use according to claim 22, characterized in that The ELISA is a double antibody sandwich ELISA or an indirect ELISA.

24. The use according to claim 23, characterized in that The ELISA is a double antibody sandwich ELISA.

25. A kit, characterized in that The kit comprises the monoclonal antibody according to any one of claims 1 to 8.

26. The kit according to claim 25, characterized in that The kit also includes other reagents for enriching, isolating and / or detecting Avibacterium paragallinarum based on ELISA or immunomagnetic bead technology.

27. The kit according to claim 26, characterized in that The ELISA is a double antibody sandwich ELISA or an indirect ELISA.

28. The kit according to claim 27, characterized in that The ELISA is a double antibody sandwich ELISA.

29. A method for enriching, isolating and / or detecting Avibacterium paragallinarum, characterized in that: The method comprises contacting a sample to be tested with the monoclonal antibody according to any one of claims 1 to 8 or the kit according to any one of claims 25 to 28 to separate, enrich and / or detect Avibacterium paragallinarum in the sample to be tested; Wherein, the method is for non-diagnostic purposes.

30. The method of claim 29, wherein: The method is an ELISA-based method.

31. The method of claim 30, wherein: The ELISA is a double antibody sandwich ELISA or an indirect ELISA.

32. The method of claim 31, wherein: The ELISA is a double antibody sandwich ELISA.

33. The method of claim 32, wherein: The double antibody sandwich ELISA is selected from one or more of the following conditions: (1) The capture antibody used in the double antibody sandwich ELISA is the monoclonal antibody, and / or the detection antibody used in the double antibody sandwich ELISA is an antibody obtained by immunizing rabbits with one or more Avibacterium paragallinarum; (2) The mass ratio of the capture antibody to the detection antibody is (2-5):1; (3) The criterion for determining a positive result by the method is: 450 When the value is ≥ mean value + 3 × standard deviation, it is judged as positive.

34. The method of claim 33, wherein: The A. paragallinarum is selected from one or more of serotype A A. paragallinarum, serotype B A. paragallinarum and serotype C A. paragallinarum.

35. The method according to claim 33 or 34, characterized in that The mass ratio of the capture antibody to the detection antibody is (2-3):1.

Images