A monoclonal antibody against Avibacterium paragallinarum, immunomagnetic beads labeled with the monoclonal antibody and application thereof

Abstract

The present invention discloses a monoclonal antibody against Avibacterium paragallinarum, immunomagnetic beads labeled therewith and applications thereof. 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 has high titer and strong specificity, and is suitable for the preparation of immunomagnetic beads based on the monoclonal antibody. The prepared immunomagnetic beads have a high capture rate, strong specificity and sensitivity for Avibacterium paragallinarum.

Description

Technical Field

[0001] The invention belongs to the technical field of pathogen separation, and particularly relates to a monoclonal antibody against Avibacterium paragallinarum, immunomagnetic beads labeled with the monoclonal antibody and applications thereof. Background Art

[0002] Infectious coryza (IC) is an acute respiratory infectious disease of chickens caused by Avibacterium paragallinarum (Apg), which is associated with decreased egg production and slow growth of laying hens. The clinical symptoms of the disease generally include runny nose, facial swelling, tearing, conjunctivitis, etc. Chickens with the disease have a high mortality rate after mixed infection with Avibacterium paragallinarum and other pathogens. The disease develops quickly and spreads rapidly, causing great economic losses to the poultry industry.

[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 use sample pretreatment methods to remove interfering substances and background signal 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. The bacterial enrichment technology can quickly concentrate the target bacteria in the sample within tens of minutes. Among the many enrichment technologies used in sample processing, the immunomagnetic bead separation technology based on antigen-antibody reaction is currently a hot research topic. This technology has many advantages, such as shortening the culture time of microbial detection, removing substances in the matrix that affect the determination, reducing the amount of sample required, etc., thereby achieving rapid enrichment of microorganisms. Especially for samples with low original concentrations, enrichment technology is conducive to improving the accuracy of analytical methods and expanding the application scope of detection technology. At the same time, it can also help improve the sampling technology required for detecting low-level pathogens or sporadic pollution. This technology has been widely used in the rapid enrichment and detection of pathogenic microorganisms.

[0004] Monoclonal antibodies (MABs) can specifically recognize and bind to their corresponding antigens. In the immunomagnetic separation technology based on antigen-antibody reaction, obtaining specific MABs is the most critical technology. MABs with high affinity and strong specificity are necessary conditions for the immunomagnetic technology to achieve high capture rate and high specificity. MABs are coupled with magnetic beads to prepare immunomagnetic beads. By further optimizing a variety of reaction conditions and under the action of a magnetic field, rapid, efficient and specific enrichment of pathogenic microorganisms in samples can be achieved, which can then be used for the separation and detection of target pathogens. Summary of the invention

[0005] In order to solve the defect that the prior art cannot achieve rapidity, simplicity, specificity and sensitivity when directly detecting Avibacterium paragallinarum in food, the present invention provides a monoclonal antibody against Avibacterium paragallinarum, immunomagnetic beads labeled with the antibody and applications thereof.

[0006] The present invention solves the above technical problems through the following technical solutions.

[0007] The first aspect of the present invention provides 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.

[0008] 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.

[0009] 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.

[0010] 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.

[0011] 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.

[0012] 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.

[0013] In one embodiment of the present invention, the monoclonal antibody is a full-length antibody.

[0014] 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.

[0015] In a specific embodiment of the present invention, the amino acid sequence of the light chain of the monoclonal antibody is shown as SEQ ID NO:2; and / or, the amino acid sequence of the heavy chain of the monoclonal antibody is shown as SEQ ID NO:8.

[0016] 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.

[0017] In one embodiment of the present invention, the nucleotide sequence of the light chain of the monoclonal antibody is as shown in SEQ ID NO:1; and / or, the nucleotide sequence of the heavy chain of the monoclonal antibody is as shown in SEQ ID NO:7.

[0018] 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.

[0019] 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.

[0020] 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.

[0021] In one embodiment of the present invention, the eukaryotic cell is a yeast cell or a mammalian cell.

[0022] A fifth aspect of the present invention provides a method for preparing monoclonal antibodies against Avibacterium paragallinarum, the method comprising the following steps:

[0023] The transformant according to the fourth aspect of the present invention is cultured, and the monoclonal antibody is obtained from the culture.

[0024] The sixth aspect of the present invention provides an antibody-coupled immunomagnetic bead, wherein the antibody is the monoclonal antibody described in the first aspect of the present invention.

[0025] In one embodiment of the present invention, in the immunomagnetic beads, the mass ratio of magnetic beads to monoclonal antibodies is 4:(1-3), preferably 4:(2-3), and more preferably 4:2.

[0026] In one embodiment of the present invention, the diameter of the magnetic beads is 180-2000 nm, preferably 750 nm.

[0027] In one embodiment of the present invention, the magnetic beads are activated magnetic beads; preferably, the activation adopts EDC solution and NHS solution; more preferably, the concentration of the EDC solution and / or NHS solution is 2-8 mg / mL, for example 5 mg / mL; and / or the volume ratio of the EDC solution and the NHS solution is 1:1-5, for example 1:1.

[0028] In one embodiment of the present invention, the coupling condition is 10-100 rpm for more than 3 hours, preferably 30 rpm for more than 3 hours.

[0029] In one embodiment of the present invention, the coupling buffer used in the coupling is PBST buffer; preferably, the concentration of the PBST buffer is preferably 0.005-0.015 M, for example, 0.01 M; and / or, the pH of the PBST buffer is 6.0-8.0, for example, 7.0.

[0030] The seventh 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, the transformant described in the fourth aspect of the present invention or the immunomagnetic beads described in the sixth aspect of the present invention in the preparation of a kit for enriching, separating and / or detecting Avibacterium paragallinarum.

[0031] In one embodiment of the present invention, the A. paragallinarum is selected from one or more of serotype A A. paragallinarum, serotype B A. paragallinarum and serotype C A. paragallinarum.

[0032] The eighth aspect of the present invention provides a kit, which comprises the monoclonal antibody as described in the first aspect of the present invention or the immunomagnetic beads as described in the sixth aspect of the present invention.

[0033] The ninth aspect of the present invention provides a method for enriching and isolating Avibacterium paragallinarum, the method comprising contacting a sample to be tested with the immunomagnetic beads described in the sixth aspect of the present invention or the kit described in the eighth aspect of the present invention, to separate and enrich Avibacterium paragallinarum in the sample to be tested.

[0034] In one embodiment of the present invention, the method is selected from one or more of the following conditions:

[0035] (1) the A. paragallinarum is selected from one or more of serotype A A. paragallinarum, serotype B A. paragallinarum, and serotype C A. paragallinarum;

[0036] (2) The contact time is 40 to 90 minutes, preferably 45 to 75 minutes;

[0037] (3) Taking the concentration of Avibacterium paragallinarum as 104 In terms of CFU / mL, the mass of the monoclonal antibody used is 0.2-0.3 mg, for example, 0.2 mg, 0.3 mg; and / or the mass of the magnetic beads used is 0.2-0.5 mg, for example, 0.4 mg.

[0038] In one embodiment of the invention, the method is for diagnostic purposes.

[0039] 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.

[0040] The reagents and raw materials used in the present invention are commercially available.

[0041] The positive and progressive effects of the present invention are:

[0042] In order to establish a rapid enrichment and detection method for Avibacterium paragallinarum in samples, this study used Avibacterium paragallinarum to immunize mice, and prepared anti-Avibacterium paragallinarum monoclonal antibodies through cell fusion screening. The antibody has strong specificity and good sensitivity. The anti-Avibacterium paragallinarum monoclonal antibody prepared by the present invention has a high titer (for example, up to 1:20,480,000), and the monoclonal antibody has strong specificity; and is suitable for the preparation of monoclonal antibody-based immunomagnetic beads, and the prepared immunomagnetic beads have a high capture rate for Avibacterium paragallinarum, up to more than 90%; the prepared immunomagnetic beads have strong specificity and no cross-reaction with other common foodborne bacteria; the prepared immunomagnetic beads have good sensitivity, and when the concentration of Avibacterium paragallinarum in the capture system is as low as 10 2 CFU / mL, the capture rate of immunomagnetic beads was still above 80%. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 The results of capturing A. paragallinarum under conditions of immunomagnetic beads of different diameters are shown.

[0044] Figure 2 The results of capturing A. paragallinarum under immunomagnetic bead conditions with different amounts of monoclonal antibodies are shown.

[0045] Figure 3 The results of capturing A. paragallinarum under different amounts of magnetic beads are shown.

[0046] Figure 4 The results of capturing A. paragallinarum under different coupling buffer conditions are shown.

[0047] Figure 5 The results of capturing A. paragallinarum under different capture time conditions are shown.

[0048] Figure 6 The results of the immunomagnetic bead specificity evaluation (A) and sensitivity evaluation (B) of A. paragallinarum are shown. DETAILED DESCRIPTION

[0049] 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:

[0050] 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.

[0051] 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.

[0052] 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.

[0053] 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.

[0054] 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.

[0055] 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.

[0056] 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.

[0057] 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.

[0058] 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.

[0059] 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.

[0060] 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.

[0061] 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.

[0062] Female blank BALB / C (SPF grade) and Kunming mice (SPF grade) were purchased from Shanghai Sibeifu Experimental Animal Co., Ltd., and SP2 / 0 myeloma cells were preserved by our laboratory.

[0063] The existing strains and reagent information involved in the following examples are shown in Table 1, Table 2 and Table 3:

[0064] Table 1. Source of strains

[0065]

[0066]

[0067] Table 2 Preparation of main reagents and culture medium

[0068]

[0069]

[0070] Table 3 Main reagents and sources

[0071]

[0072]

[0073] Example 1 Preparation of Avibacterium paragallinarum Immunogen

[0074] (1) Preparation of Avibacterium paragallinarum immunogen:

[0075] a. Bacterial liquid amplification: Inoculate 3 bacterial strains, CVCC 254, CVCC257 and CVCC256, into 10 mL TSB medium and amplify to OD 600The culture medium was placed in a 37°C, 5% CO2 incubator for 20-24 hours and the growth characteristics were observed.

[0076] b. Inactivation of bacterial solution: amplify 10mL of bacterial solution, and when OD 600 When the concentration of CVCC 254, CVCC257 and CVCC256 was 1, the final concentration of 0.3% formaldehyde was used for inactivation for more than 4 hours. 1 mL was taken and centrifuged at 12000 rpm for 5 minutes. The formaldehyde was removed by washing 5 times with PBS. The bacterial suspensions of CVCC 254, CVCC257 and CVCC256 were mixed in equal amounts and adjusted to 5.0×10 7 CFU / mL and place at 4℃ for later use.

[0077] 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.

[0078] (2) Animal immunization procedures and serum titer determination.

[0079] Example 2 Animal Immunization Program

[0080] The first immunization was with bacterial solution and an equal amount of Freund's complete adjuvant, and the second to fourth immunizations were with bacterial solution and an equal amount of Freund's incomplete adjuvant, and mice were immunized after emulsification for 120 seconds. When boosting intraperitoneal immunization, the bacterial solution was diluted with physiological saline, and the immunization procedure of mice is shown in Table 4.

[0081] Table 4 Mouse immunization program

[0082]

[0083] Example 3 Animal Serum Titer Determination

[0084] After booster immunization of Balb / C mice, mouse sera were collected and the concentration was 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 diluting the volume ratio of 1:1, 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.

[0085] Indirect ELISA experimental steps:

[0086] After the second 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:

[0087] a. Inoculate 3 bacterial strains, CVCC 254, CVCC257 and CVCC256, into 10 mL TSB medium and amplify until OD 600 After 1, perform formaldehyde inactivation for at least 4 hours, aspirate 100 μL and transfer to 1 mL TSB liquid culture medium, place it in a shaking incubator at 37°C and 180 rpm for 18-24 hours, and observe whether the bacterial solution is turbid.

[0088] b. Wash the inactivated 3 strains of Avibacterium paragallinarum 5 times with PBS. Dilute the bacterial solution 10-fold to 10 with ELISA coating solution. 6 CFU / mL, add 100μL of diluent to each well of a 96-well plate, and coat the three bacterial strains CVCC 254, CVCC257, and CVCC256 separately at 4°C overnight. Wash 5 times with PBST, add gelatin to block at 37°C for at least 2h. Wash 5 times with PBST, dilute mouse serum in a 2-fold gradient with 5% FBS in PBST, add 100μL of diluted serum to each well of the 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, as shown in Table 5 below.

[0089] Table 5 ELISA test results of serum levels in mice after immunization

[0090]

[0091] Example 4 Preparation of Avibacterium paragallinarum monoclonal antibody

[0092] (1) Cultivation of myeloma cells (SP2 / 0)

[0093] 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.

[0094] 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.

[0095] (2) Preparation of feeder cells

[0096] 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.

[0097] b. Kill Kunming mice from the eyeball, 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 upward 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.

[0098] 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.

[0099] 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 culture medium containing 20% ​​FBS HAT.

[0100] 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.

[0101] (3) Cell fusion

[0102] 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.

[0103] 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.

[0104] 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.

[0105] 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.

[0106] 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.

[0107] 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.

[0108] 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.

[0109] h. Add 15 mL of preheated blank DMEM, let stand for 10 min, centrifuge at 1000 rpm for 8 min, and discard the supernatant.

[0110] 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.

[0111] (4) Screening of positive clones after fusion

[0112] 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.

[0113] b. ELISA steps were the same as those in Example 3, with Avibacterium paragallinarum JZIC008, JZIC026 and JZIC043 mixed and coated. The primary antibody was 90 μL of PBST containing 5% FBS and 10 μL of cell supernatant, and the remaining steps were the same as those in step (b).

[0114] 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 pH is 1.0, add formaldehyde, inactivate at least 4 hours at 37°C, 180 rpm 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 multiples, and use them as the primary antibody. The rest of the steps are the same as step (b) of Example 3.

[0115] (5) Subcloning of positive hybridoma cells

[0116] a. Pick monoclonal cell lines with high titer of cell supernatant and good growth status for subcloning. First, mark the cell position of positive clones under a microscope, use a 200μL pipette to absorb a small amount of cells and transfer them to a 96-well cell plate, and dilute them in a gradient with an incomplete medium of 20% FBS. Observe under an inverted microscope, select wells containing 90-120 cells per well, transfer the liquid in the well to 10mL of incomplete medium of 20% FBS, and mix them by blowing. Then add it dropwise to a 96-well cell plate containing feeder cells (100μL / well) and culture it in a 37℃, 5% CO2 incubator. Observe the cell growth status in time and supplement the culture medium appropriately 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 of the monoclonal well reaches 100%, expand the culture, freeze and mark it. b. On the 3rd-4th day after cell fusion, examine all cell culture wells under a microscope, and mark the 96 wells with 1-2 monoclonal clones. On the 8th to 9th day after cell fusion, the culture supernatant containing monoclonal cells was selected for titer detection. Subsequently, monoclonal cell lines with high titer and good specificity were selected for subcloning.

[0117] (6) Preparation of mouse ascites

[0118] 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.

[0119] (7) Purification of monoclonal antibodies

[0120] Mouse ascites was diluted 10-20 times with PBS (pH7.4), mixed, and filtered with a 0.22 pore filter. Wash the magnetic beads with 10mL of PBS (pH7.4), add 1mL of PBS (pH7.4) each time to mix the magnetic beads, centrifuge, let stand for 1min, and discard the supernatant; add the magnetic beads to the diluted ascites above, and bind at 4℃ for 2-3h. Take out, centrifuge, place on a magnetic stand, let stand for 5min, collect the supernatant, draw 5mL of eluent (0.1M Gly-HCl, pH2.7) buffer, add 900μL to the magnetic beads each time, mix, centrifuge, let stand for 1min, draw the supernatant, add 100μL of neutralizing solution (0.5M Tris-Cl, pH8.0) to neutralize, prepare 5 1mL centrifuge tubes, and take multiple tubes in sequence when taking over, and record the order. Measure the antibody concentration, which is 1.95 mg / mL, and store at -20°C for later use. After elution, wash the balanced magnetic beads with at least 10 mL of PBS. Store the magnetic beads in a storage solution (PBS containing 20% ​​ethanol) at 4°C.

[0121] Example 5 Monoclonal Antibody Analysis

[0122] 1. Potency and specificity determination

[0123] The titer of the A. paragallinarum-specific monoclonal antibody was measured by ELISA. The inactivated bacterial solutions of 3 strains of A. paragallinarum CVCC 254, CVCC257 and CVCC256 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 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 the positive / negative (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 180rpm in a constant temperature shaker until the bacterial solution concentration was OD 600 When the concentration was 1.0, formaldehyde was added and the cells were inactivated at 37°C, 180 rpm constant temperature shaker for at least 4 hours, and washed with PBS 5 times. The cells were resuspended in the coating solution as the coating source. The primary antibody was 10 μL mouse ascites + 90 μL 5% serum PBST. The secondary antibody concentration was 1:10000, and the remaining steps were the same as step (b) of Example 3.

[0124] After 4 subclonings, a monoclonal antibody was screened out and named 1-E10. The titer of ascites was determined by indirect ELISA using Avibacterium paragallinarum as the coating source. The specific method was the same as above. From the results (Table 6), it can be seen that the titer of monoclonal antibody 1-E10 is relatively high. When diluted 5120000 times, the OD 450 It was still above 0.6, and the P / N value was ≥2.1, so the titer of 1-E10 was 1:20,480,000. Inactivated Bacillus subtilis, Streptococcus uberis, Clostridium perfringens, Escherichia coli O157, Staphylococcus aureus, Proteus and Salmonella pullorum were used as coating sources, and 1-E10 was diluted 50,000 times to measure the specificity of the antibody. The results (Table 7) showed that none of the antibodies reacted with the above 7 common foodborne pathogens, indicating that the antibody specificity was good.

[0125] Table 6 OD of monoclonal antibodies 450 Determination

[0126] Dilution multiple <![CDATA[OD 450 ]]> 1:10,00 2.996 1:20,00 2.812 1:40,00 2.511 1:80,00 2.236 1:160,00 1.951 1:320,00 1.64 1:640,00 1.314 1:1,280,00 1.106 1:2,560,00 0.964 1:5,120,00 0.624 Negative 0.054

[0127] Table 7 Specificity determination of monoclonal antibodies

[0128]

[0129]

[0130] 2. Identification of monoclonal antibodies

[0131] The 96-well ELISA plate was coated with inactivated Avibacterium paragallinarum, and the antibody subtype of the collected 1 positive antibody was identified using a monoclonal antibody subtype identification kit. The primary antibody was diluted at 1:10,000, and the secondary antibody was added at a dilution of 1:300 using the antibody in the kit.

[0132] The subtype of the above monoclonal antibody was identified using an antibody subtype identification kit. The results showed that the light chain type of monoclonal antibody 1-E10 was kappa type and the heavy chain type was IgG2a type.

[0133] Example 6 Determination and analysis of anti-Avibacterium paragallinarum monoclonal antibody sequences

[0134] In order to identify the gene sequence of monoclonal antibody 1-E10, total RNA was isolated from hybridoma cells secreting monoclonal antibody 1-E10 using Trizol reagent. Specific steps: (1) Trizol lysis: resuspend all bacteria with 1 mL Trizol, mix well by pipetting and let stand for 3-5 min. (2) Chloroform phase separation: add 0.2 mL of chloroform (trichloromethane) to each sample, shake vigorously for 15 s, let it stand at room temperature for 3 min, and then centrifuge at 12,000 rpm for 15 min. (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 well 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 min. (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 .

[0135] 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.

[0136] In order to determine the antibody sequence, RNA was extracted from hybridoma cells, and then the variable region sequence was amplified by degenerate primer method. The sequencing results showed that the sequence of the variable region of the monoclonal antibody was a gene fragment of the germline gene family (Table 8).

[0137] Table 8 Germline genes of variable regions of anti-Avibacterium paragallinarum monoclonal antibodies (IgBlast analysis)

[0138] MAb <![CDATA[V H ]]> <![CDATA[D H ]]> <![CDATA[J H ]]> <![CDATA[V K ]]> <![CDATA[J K ]]> 1-E10 12-1*01 2-12*01 4*01 4-59*01 5*01

[0139] 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.

[0140] The sequencing results are as follows:

[0141] Light chain nucleotide sequence:

[0142] CAAATTGTTCTCACCCAGTCTCCAACAATCATGTCTGCATCTCCAGGGGAGAAGGTCACCATGACCTGCAGTGCCAGCTCAAGTGTACGTTACATGCACTGGTACCAGCAGAAGTCAGGCACCTCCCCCAAAAGATGGATTTATGAAACATCAAAACTGGCTTCTGGAGTCCCTGCTCGCTTCAGTGGCAGTGGGTCTGGGACCTCTTACTCTCTCACAATCAGCAGCATGGAGGCTGAAGATGCTGCCACTTATTACTGCCAGCAGTGGAGTAGTAACCCGCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTCAAACGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGTTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTATGAACGACATAACAGCTATACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTGTTAG(SEQ ID NO:1)

[0143] Note: The bold part is the nucleotide sequence of the variable region, and the non-bold part is the nucleotide sequence of the constant region.

[0144] Amino acid sequence of the light chain:

[0145] QIVLTQSPTIMSASPGEKVTMTCSAS SSVRY MHWYQQKSGTSPKRWIY ETSKLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC (SEQ ID NO: 2) 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 complementary determining regions (CDRs).

[0146] in:

[0147] LCDR1:SSVRY (SEQ ID NO:3)

[0148] LCDR2: ETS (SEQ ID NO: 4)

[0149] LCDR3: QQWSSNPLT(SEQ ID NO:5)

[0150] Light chain variable region amino acid sequence:

[0151] QIVLTQSPTIMSASPGEKVTMTCSASSSVRYMHWYQQKSGTSPKRWIYETSKLAS GVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELK(SEQ ID NO:6)

[0152] Heavy chain nucleotide sequence:

[0153]

[0154] Note: The bold part is the variable region nucleotide sequence, and the non-bold part is the constant region nucleotide sequence.

[0155] Heavy chain amino acid sequence:

[0156] EVQLVESGGGLVKPGGSLKLSCAAS GFLFSSYD MSWVRQTPEKRLEWVAK ISGSGSKT HYPDTVKGRFTISRDNAKNTLFLQLSSLQSEDTAMYYC TRHSYRYVMDY WGQGTSVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDD VEVHTAQTQPREEQFNSTFRSSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK(SEQ IDNO:8)

[0157] 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).

[0158] in:

[0159] HCDR1: GFLFSEVQ (SEQ ID NO: 9)

[0160] HCDR2:ISGSGSKT(SEQ ID NO:10)

[0161] HCDR3:TRHSYRYVMDY(SEQ ID NO:11)

[0162] Heavy chain variable region amino acid sequence:

[0163] EVQLVESGGGLVKPGGSLKLSCAASGFLFSSYDMSWVRQTPEKRLEWVAKISGSG SKTHYPDTVKGRFTISRDNAKNTLFLQLSSLQSEDTAMYYCTRHSYRYVMDYWGQGT SVTVSS(SEQ ID NO:12)

[0164] Example 7 Preparation of Avibacterium paragallinarum Immunomagnetic Beads

[0165] The steps for preparing the Avibacterium paragallinarum immunomagnetic beads are as follows:

[0166] a. Mix the magnetic beads, pipette 100 μL of magnetic beads (10 mg / mL) into an EP tube, place the EP tube on a magnetic rack and let it stand for 1 min, discard the supernatant, pipette 500 μL of activation buffer (MEST, pH 6.0) to resuspend the magnetic beads, and repeat this step 3 times.

[0167] b. Take 100 μL of 5 mg / mL EDC solution and NHS solution (pre-prepared) into an EP tube, mix well, and place in a constant temperature shaker at 37°C and 180 rpm for activation for 30 min.

[0168] c. After 30 minutes, take out the EP tube, centrifuge it, place the EP tube on a magnetic rack, discard the supernatant, mix it with 500 μL buffer (MEST, pH 6.0), place it on a magnetic rack, let it stand for 1 minute, discard the supernatant, and repeat three times.

[0169] d. Take a new EP tube, draw 300 μg of the Avibacterium paragallinarum antibody prepared in Example 4 and add it to 500 μL of coupling buffer, mix well, add it to the activated magnetic beads, place it on a rotating incubator, and couple it at 30 rpm for more than 3 hours.

[0170] e. Take out the EP tube, spin it gently, place it on a magnetic rack for 1 min, wash it 4 times with 500 μL PBST, add 1 mL of blocking solution, place it on a 37°C shaker, and block it at 180 rpm for more than 30 min.

[0171] f. Take out the EP tube and spin gently, place the EP tube on a magnetic rack, wash with PBST 4 times, discard the supernatant, add 0.6mL-1mL storage solution, resuspend the magnetic beads, and store in a 4℃ refrigerator for later use.

[0172] g. Inoculate Avibacterium paragallinarum into TSB liquid culture medium and place in a shaker at 37°C, 180 rpm. After culturing for 18-24 hours, when OD 600 When the concentration is 0.5, dilute the bacterial solution 10-fold with PBST to 10 4 CFU / mL.

[0173] h. Take a new 1.5mL EP tube and add 100μL of magnetic beads, discard the supernatant, add 100μL of fresh bacterial solution and 900μL of PBST, a total of 1mL, mix gently, place on a rotator at 30rpm, and react at room temperature for 45min.

[0174] i. After capture, gently spin off, place on a magnetic stand, and let stand for 1 minute. Aspirate 100μL of the supernatant and apply it to a TSA agar plate. Wash the magnetic beads once with 500μL PBST, discard the supernatant, aspirate 100μL PBST to resuspend the magnetic beads, and apply all of them to a TSA agar plate. Mark, place in a 37℃ incubator, and after 18-24 hours of incubation, take out the plate for colony counting and use 10 3 Bacterial solution was used as the control group.

[0175] j. Calculate the capture rate. The capture rate is the number of magnetic bead colonies / (the number of supernatant colonies + the number of magnetic bead colonies).

[0176] Example 8 Optimization of immunomagnetic bead detection conditions

[0177] 1. Determination of the Optimal Diameter of Immunomagnetic Beads

[0178] According to previous studies, changes in the diameter of immunomagnetic beads can have a significant impact on the capture of specific bacteria. In this experiment, Avibacterium paragallinarum CVCC257 was used as the target bacterium, and PBST was gradiently diluted to about 10 4 CFU / mL, four groups of magnetic beads with different diameters (PM3-020 (diameter 180nm), PM3-050 (diameter 750nm), SM3-100 (diameter 1150nm), PM3-100 (diameter 2μm)) were selected to optimize the diameter of the magnetic beads, and the experimental method was the same as Example 7. This experiment was repeated 3 times, the capture rate was calculated, and the optimal diameter of the immunomagnetic beads was determined.

[0179] like Figure 1 As shown in the figure, the immunomagnetic beads prepared by magnetic beads of different particle sizes have different effects. Taking Avibacterium paragallinarum CVCC257 as the target strain, four groups of magnetic beads with different diameters were selected for magnetic bead diameter optimization. Among the four types of PM3-050 (750nm), PM3-020 (180nm), SM3-D100 (1150nm) and PM3-100 (2μm) magnetic beads, the magnetic beads with the highest capture rate were PM3-050 (750nm) with a capture rate of 96.7%, followed by SM3-D100 (1150nm) with a capture rate of 74.15%. The capture rate of PM3-050 (750nm) was significantly higher than that of SM3-D100 (1150nm) (P<0.01), and magnetic beads with a diameter of 750nm were selected to continue the subsequent experiments.

[0180] 2. Determination of the optimal antibody workload

[0181] A diameter of 750 nm was selected as the diameter of the magnetic beads. 0.4 mg of the activated magnetic beads were taken and coupled with 0.1 mg, 0.2 mg and 0.3 mg of monoclonal antibodies respectively. The other steps of the experiment were the same as those in Example 7, and the experiment was repeated 3 times to calculate the capture rate and determine the optimal antibody workload.

[0182] like Figure 2 As shown in the data, when the amount of monoclonal antibody was 0.1 mg, 0.2 mg and 0.3 mg, the corresponding coupling rates of magnetic beads and monoclonal antibody were 87.3%, 94.3% and 94.6%, respectively. Among them, the capture rate of magnetic beads when 0.2 mg and 0.3 mg monoclonal antibody were coupled was significantly higher than that of magnetic beads coupled with 0.1 mg monoclonal antibody (P<0.01). Considering the saving of monoclonal antibody, 0.2 mg antibody amount was selected for the subsequent condition optimization.

[0183] 3. Determination of the Optimal Number of Immunomagnetic Beads

[0184] To explore the effect of the amount of magnetic beads used on the capture rate, Avibacterium paragallinarum CVCC257 was used as the target bacterium and the PBST gradient was diluted to about 10 4 CFU / mL, using immunomagnetic beads for capture experiments. The optimal working amount of immunomagnetic beads was determined by adding 0.1, 0.2, 0.3, 0.4 and 0.5 mg of immunomagnetic beads to each tube in turn. The experiment was repeated 3 times, the capture rate was calculated, and the optimal number of immunomagnetic beads was determined.

[0185] like Figure 3 As shown in the figure, when the dosage is less than 0.2 mg, the capture rate increases with the increase of magnetic bead dosage; when the magnetic bead dosage exceeds 0.2 mg, the capture rate tends to increase slowly. In order to achieve the maximum capture rate, when the concentration of Avibacterium paragallinarum CVCC257 is about 10 4 CFU / mL, when the dosage of magnetic beads is 0.4 mg, the magnetic bead capture rate can reach more than 95%, which is the optimal dosage.

[0186] 4. Optimization of the best coupling buffer for immunomagnetic beads

[0187] The strain Avibacterium paragallinarum CVCC257 was used as the target bacteria, and four coupling buffers, MEST (0.025M, pH 7.0), MES (0.025M, pH 6.0), PBST (0.01M, pH 7.0) and PBS (0.01M, pH 7.0), were selected, and the immunomagnetic beads were prepared with PM3-050 (diameter 750nm) magnetic beads. The other steps of the experiment were the same as those in Example 7. The experiment was repeated 3 times, the capture rate was calculated, and the optimal coupling buffer was determined.

[0188] like Figure 4As shown, when PBST (0.01M, pH 7.0) was used as the coupling buffer, the capture rate was about 97%, which was significantly higher than that of PBS, MES and MEST (P<0.01). Therefore, this experiment selected PBST (0.01M, pH 7.0) as the coupling buffer for subsequent condition optimization.

[0189] 5. Determination of the Optimal Capture Time of Immunomagnetic Beads

[0190] To explore the effect of different capture time on the capture rate, Avibacterium paragallinarum CVCC257 was used as the target bacterium and PBST was gradiently diluted to about 10 4 CFU / mL, the following four capture time groups were selected: 30min, 45min, 60min and 75min, and the capture experiment was carried out using immunomagnetic beads to determine the optimal capture time. The experiment was repeated 3 times.

[0191] like Figure 5 As shown, when the capture time was 30min, 45min, 60min and 75min, the capture rates of Avibacterium paragallinarum were 86.6%, 95.4%, 94.2% and 94.9% respectively. The plateau period was reached when the capture time was 45min. Even with the increase of capture time, the capture rate did not change significantly. Therefore, the capture time of 45min was selected for subsequent experiments.

[0192] 6. Specificity detection of immunomagnetic bead detection method

[0193] Seven other common bacteria, including Bacillus subtilis, Enterohemorrhagic Escherichia coli O157, Salmonella pullorum, Staphylococcus aureus, Clostridium perfringens, Proteus vulgaris and Streptococcus uberis, were cultured as captured strains to analyze the specificity of the immunomagnetic beads, and the experiment was repeated three times.

[0194] like Figure 6 As shown in A, specificity tests were carried out using three strains of A. paragallinarum (CVCC 254, CVCC257 and CVCC256) and seven strains (Bacillus subtilis, Streptococcus uberis, Clostridium perfringens, Escherichia coli O157, Staphylococcus aureus, Proteus and Salmonella pullorum) of other pathogens. The capture rates of the prepared immunomagnetic beads for the three strains of A. paragallinarum were all above 90%, but the capture rates for the seven pathogens were all below 25%, and the difference was extremely significant (p<0.01), indicating that the immunomagnetic beads coupled with the A. paragallinarum monoclonal antibody 1-E10 had good specificity.

[0195] 7. Sensitivity Determination of Immunomagnetic Bead Detection Method

[0196] To explore the effect of different bacterial concentrations on the capture rate, we selected A. paragallinarum CVCC257 as the target bacterium and used PBST to dilute the A. paragallinarum bacterial solution to a bacterial content of 10 in 1 mL of the capture system. 4 CFU / mL, 10 3 CFU / mL, 10 2 CFU / mL and 10 CFU / mL were used for capture, and the capture time was 45 min. The experimental steps were the same as above, and the experiment was repeated 3 times to determine the sensitivity of the immunomagnetic beads.

[0197] like Figure 6 As shown in B, the capture rate of magnetic beads with different bacterial amounts was above 80%, 4 CFU, 10 3 There was no significant change in the capture rate of CFU (p>0.05), indicating that the immunomagnetic beads had high sensitivity.

[0198] 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.

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 has at least 85% sequence identity with SEQ ID NO: 6; And / or, the amino acid sequence of the heavy chain variable region has at least 85% sequence identity with SEQ ID NO:

12.

4. The monoclonal antibody according to claim 3, characterized in that The amino acid sequence of the light chain variable region is shown in SEQ ID NO: 6; And / or, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:

12.

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

6. The monoclonal antibody according to claim 5, 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 antibody is derived from a murine heavy chain IgG2a constant region or a variant thereof, and the light chain constant region is derived from a murine light chain κ chain constant region or a variant thereof.

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. A recombinant expression vector, characterized in that: The recombinant expression vector comprises the isolated nucleic acid of claim 9.

11. The recombinant expression vector according to claim 10, characterized in that The recombinant expression vector is a plasmid, a cosmid or a viral vector.

12. The recombinant expression vector according to claim 11, characterized in that The viral vector is a bacteriophage, a retroviral vector, a lentiviral vector, an adenoviral vector or an adeno-associated viral vector.

13. A transformant, characterized in that: The transformant comprises the isolated nucleic acid according to claim 9 or the recombinant expression vector according to any one of claims 10 to 12, and the host cell of the transformant is a prokaryotic cell or a eukaryotic cell, and the host cell is a non-plant cell.

14. The transformant according to claim 13, characterized in that The eukaryotic cell is a yeast cell or a mammalian cell.

15. A method for preparing monoclonal antibodies against Avibacterium paragallinarum, characterized in that: The method comprises the following steps: The transformant according to claim 13 or 14 is cultured, and the monoclonal antibody is obtained from the culture.

16. An antibody-coupled immunomagnetic bead, characterized in that: The antibody is a monoclonal antibody according to any one of claims 1 to 8.

17. The immunomagnetic beads according to claim 16, wherein In the immunomagnetic beads, the mass ratio of magnetic beads to monoclonal antibodies is 4:(1-3); And / or, the diameter of the magnetic beads is 180-2000 nm; And / or, the magnetic beads are activated magnetic beads; And / or, the coupling condition is 10-100 rpm for more than 3 hours; And / or, the coupling buffer used in the coupling is PBST buffer.

18. The immunomagnetic beads according to claim 17, characterized in that In the immunomagnetic beads, the mass ratio of the magnetic beads to the monoclonal antibodies is 4:(2~3).

19. The immunomagnetic beads according to claim 18, characterized in that In the immunomagnetic beads, the mass ratio of magnetic beads to monoclonal antibodies is 4:

2.

20. The immunomagnetic beads according to claim 17, wherein The diameter of the magnetic beads is 750 nm.

21. The immunomagnetic beads according to claim 17, wherein The activation adopts EDC solution and NHS solution.

22. The immunomagnetic beads according to claim 21, characterized in that The concentration of the EDC solution and / or the NHS solution is 2-8 mg / mL; and / or the volume ratio of the EDC solution to the NHS solution is 1:1-5.

23. The immunomagnetic beads according to claim 22, characterized in that The concentration of the EDC solution and / or NHS solution is 5 mg / mL.

24. The immunomagnetic beads according to claim 22, characterized in that The volume ratio of the EDC solution to the NHS solution is 1:

1.

25. The immunomagnetic beads according to claim 17, wherein The coupling condition is 30 rpm for more than 3 hours.

26. The immunomagnetic beads according to claim 17, characterized in that The concentration of the PBST buffer is 0.005-0.015 M; and / or the pH of the PBST buffer is 6.0-8.

0.

27. The immunomagnetic beads according to claim 26, wherein The concentration of the PBST buffer is 0.01M.

28. The immunomagnetic bead according to any one of claims 17 to 27, characterized in that The pH of the PBST buffer is 7.

0.

29. Use of the monoclonal antibody according to any one of claims 1 to 8, the isolated nucleic acid according to claim 9, the recombinant expression vector according to any one of claims 10 to 12, the transformant according to claim 13 or 14, or the immunomagnetic beads according to any one of claims 16 to 28 in the preparation of a kit for enriching, separating and / or detecting Avibacterium paragallinarum.

30. The use according to claim 29, characterized in that The A. paragallinarum is selected from one or more of serotype A A. paragallinarum, serotype B A. paragallinarum and serotype C A. paragallinarum.

31. A kit, characterized in that The kit comprises the monoclonal antibody according to any one of claims 1 to 8 or the immunomagnetic beads according to any one of claims 16 to 28.

32. A method for enriching and isolating Avibacterium paragallinarum, characterized in that: The method comprises contacting the immunomagnetic beads according to any one of claims 16 to 28 or the kit according to claim 31 with a sample to be tested, and isolating and enriching Avibacterium paragallinarum in the sample to be tested; wherein the method is for non-diagnostic purposes.

33. The method of claim 32, wherein: The method is selected from one or more of the following conditions: (1) the A. paragallinarum bacterium is selected from one or more of serotype A A. paragallinarum, serotype B A. paragallinarum, and serotype C A. paragallinarum; (2) The contact time is 40 to 90 minutes; (3) Taking the concentration of Avibacterium paragallinarum as 10 4 In terms of CFU / mL, the mass of the monoclonal antibody used is 0.2-0.3 mg; and / or the mass of the magnetic beads used is 0.2-0.5 mg.

34. The method of claim 33, wherein: The contact time is 45 min to 75 min.

35. The method of claim 33, wherein: The usage mass of the monoclonal antibody is 0.2 mg or 0.3 mg.

36. The method according to any one of claims 33 to 35, characterized in that The mass of the magnetic beads used is 0.4 mg.

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