Antibodies to campylobacter-specific proteins, antibody-coupled magnetic beads and uses thereof

Abstract

The application discloses a Campylobacter-specific protein antibody, an antibody-coupled magnetic bead and application. The antibody is a monoclonal antibody 1A10 and a monoclonal antibody 3D9, the monoclonal antibody 1A10 comprises a light chain variable region and a heavy chain variable region, the amino acid sequences of the light chain variable region and the heavy chain variable region of the monoclonal antibody 1A10 are shown in SEQ ID NO: 7 and 8 respectively; the monoclonal antibody 3D9 comprises a light chain variable region and a heavy chain variable region, the amino acid sequences of the light chain variable region and the heavy chain variable region of the monoclonal antibody 3D9 are shown in SEQ ID NO: 9 and 10 respectively. The antibody-coupled magnetic bead is coupled by the monoclonal antibody 1A10, the monoclonal antibody 3D9 and activated carboxyl magnetic beads respectively. The antibody-coupled magnetic bead can specifically enrich Campylobacter, has high enrichment efficiency, simple enrichment operation, can be applied to metabolic product analysis of Campylobacter, and can be used for qualitative and quantitative analysis of the Campylobacter.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to an antibody against a Campylobacter genus-specific protein, antibody-conjugated magnetic beads, and their applications. Background Technology

[0002] Campylobacter was first discovered in 1963. It is comma-shaped or S-shaped, microaerophilic, possesses extreme flagella, exhibits rapid motility, does not form spores, and is Gram-negative. The bacterial cell size is (0.2–0.5) × (1.5–5) micrometers, with longer cells exhibiting 4–5 bends. It grows readily on blood agar, forming both smooth and rough colonies. The five most common species in the Campylobacter genus are Campylobacter jejuni, Campylobacter coli, Campylobacter fetus, Campylobacter guillier, and Campylobacter embryonicum. Campylobacter fetus subsp. jejuni is a significant pathogen causing acute diarrhea in infants and young children in many developing countries, and some Campylobacter species can cause abortion in cattle and sheep.

[0003] Extracting specific natural microorganisms or groups of microorganisms from the feces of healthy individuals or patients plays a significant role in elucidating the relationship between microorganisms and diseases, and in improving intestinal microbiota transplantation techniques. Traditional microbial isolation typically employs selective culture media, using plates to screen for desired strains. However, this method relies heavily on the culture medium; some microorganisms cannot be cultured and therefore cannot be obtained, while others become weaker and are eliminated during the culturing process, making them difficult to acquire.

[0004] Current reports on detection methods for Campylobacter include immunological and nucleic acid-based methods, as well as methods for preparing Campylobacter jejuni antibodies by selecting a single target protein. One method involves tandemly selecting two different target protein fragments of Campylobacter, followed by recombinant expression in Escherichia coli. This fusion protein can be used to screen for antibodies targeting two different targets in a single cell fusion, but methods for preparing antibodies using this method have not yet been reported. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of existing technologies and provide an antibody, antibody-conjugated magnetic beads, and applications for Campylobacter spp. specific proteins. This invention involves immunizing mice with the fusion protein CadF-CDTB, preparing monoclonal antibodies 1A10 and 3D9 using hybridoma technology, and then conjugating the monoclonal antibodies 1A10 and 3D9 with magnetic beads. Campylobacter spp. are then enriched based on the antibody-conjugated magnetic beads. The antibody-conjugated magnetic beads of this invention capture Campylobacter spp. with high specificity and sensitivity, and can improve enrichment efficiency. It can be applied to the analysis of Campylobacter spp. metabolites, as well as for their qualitative and quantitative analysis.

[0006] To achieve the above objectives, the technical solution designed by the present invention is as follows:

[0007] This invention provides a monoclonal antibody against a Campylobacter genus-specific protein, wherein the monoclonal antibody comprises monoclonal antibody 1A10 and monoclonal antibody 3D9;

[0008] Monoclonal antibody 1A10 includes a light chain variable region and a heavy chain variable region. The amino acid sequence of the light chain variable region of monoclonal antibody 1A10 is shown in SEQ ID NO: 7, and the amino acid sequence of the heavy chain variable region of monoclonal antibody 1A10 is shown in SEQ ID NO: 8.

[0009] Monoclonal antibody 3D9 includes a light chain variable region and a heavy chain variable region. The amino acid sequence of the light chain variable region of monoclonal antibody 3D9 is shown in SEQ ID NO: 9, and the amino acid sequence of the heavy chain variable region of monoclonal antibody 3D9 is shown in SEQ ID NO: 10.

[0010] Furthermore, the light chain variable region of the monoclonal antibody 1A10 includes three complementarity-determining regions, namely:

[0011] 1A10-CDR-L1:HGWRWPDCWACWNFR;

[0012] 1A10-CDR-L2: APWCADW;

[0013] 1A10-CDR-L3: SRVH;

[0014] The heavy chain variable region of monoclonal antibody 1A10 includes three complementarity-determining regions, namely:

[0015] 1A10-CDR-H1: ENCPE;

[0016] 1A10-CDR-H2:EVCFCCAACVNCSRPHA;

[0017] 1A10-CDR-H3: KGLWFASAPENS;

[0018] The light chain variable region of monoclonal antibody 3D9 includes three complementarity-determining regions, namely:

[0019] 3D9-CDR-L1: HWWSTAIRWCACQPAR;

[0020] 3D9-CDR-L2: RPWCHPW;

[0021] 3D9-CDR-L3: WTWCKPKCN;

[0022] The heavy chain variable region of monoclonal antibody 3D9 includes three complementarity-determining regions, namely:

[0023] 3D9-CDR-H1: SCNFW;

[0024] 3D9-CDR-H2:KVEFGVAENSWEFRPRW;

[0025] 3D9-CDR-H3: PFANSQT.

[0026] Furthermore, the monoclonal antibody 1A10 and monoclonal antibody 3D9 are prepared from hybridoma cell lines.

[0027] This invention also provides a method for preparing a hybridoma cell line, comprising the following steps:

[0028] (1) The nucleotide sequences of the codon-optimized proteins CadF and CDTB were fused to obtain the fusion protein CadF-CDTB;

[0029] (2) The nucleotide sequence of the fusion protein CadF-CDTB was transformed into Escherichia coli BL21, expressed, and purified by sonication to obtain the purified protein;

[0030] (3) Mix the purified protein with Freund's adjuvant, emulsify it and then immunize the mice;

[0031] (4) Then, spleen cells from immunized mice were fused with myeloma cells SP2 / 0, and hybridoma cell lines 1A10 and 3D9 were obtained through screening.

[0032] Furthermore, the amino acid sequence of the protein CadF is shown in SEQ ID NO: 4, the amino acid sequence of the protein CDTB is shown in SEQ ID NO: 6, and the amino acid sequence of the fusion protein CadF-CDTB is shown in SEQ ID NO: 2.

[0033] Furthermore, the optimized nucleotide sequence of the protein CadF codon is shown in SEQ ID NO: 3, the optimized nucleotide sequence of the protein CDTB codon is shown in SEQ ID NO: 5, and the nucleotide sequence of the fusion protein CadF-CDTB is shown in SEQ ID NO: 1.

[0034] The present invention also provides the application of the monoclonal antibody in the preparation of antibody-conjugated magnetic beads.

[0035] This invention also provides a method for preparing antibody-conjugated magnetic beads, comprising the following steps:

[0036] (1) Dilute monoclonal antibody 1A10 and monoclonal antibody 3D9 to 1.5-2.5 mg / mL using morpholine ethanesulfonic acid buffer to obtain diluted monoclonal antibody 1A10 and monoclonal antibody 3D9;

[0037] (2) The magnetic beads were activated by carboxyl groups to obtain an activated carboxyl magnetic bead solution;

[0038] (3) The diluted monoclonal antibody 1A10 and monoclonal antibody 3D9 were coupled with the activated carboxyl magnetic bead solution to obtain antibody-coupled magnetic beads 1A10 and antibody-coupled magnetic beads 3D9, wherein the particle size of the activated carboxyl magnetic beads was 10-30 μm, and the molar ratio of monoclonal antibody 1A10 or monoclonal antibody 3D9 to the activated carboxyl magnetic bead solution was 1:5-10.

[0039] Further, the monoclonal antibody 1A10 and monoclonal antibody 3D9 are diluted to 2 mg / mL;

[0040] The activated carboxyl magnetic beads have a particle size of 10 μm, and the molar ratio of monoclonal antibody 1A10 or monoclonal antibody 3D9 to the activated carboxyl magnetic bead solution is 1:5.

[0041] The present invention also provides the application of antibody-conjugated magnetic beads prepared by the above preparation method in enriching Campylobacter spp., wherein the antibody-conjugated magnetic beads are one or two of antibody-conjugated magnetic beads 1A10 and antibody-conjugated magnetic beads 3D9.

[0042] The principle of this invention:

[0043] 1. Studies on Campylobacter intestinal colonization and virulence include flagella-mediated motility, bacterial adhesion to the intestinal mucosa, invasiveness, and toxin production. Important virulence determinants include the CadF protein (a 37kDa fibronectin-binding outer membrane protein), responsible for Campylobacter adhesion to fibronectin. The CadF protein selected in this invention shares over 90% homology with 45 other species of the Campylobacter genus and can serve as a target protein for recognizing Campylobacter. Another characteristic toxin is the cell swelling toxin, composed of three subunits: CDTA, CDTB, and CDTC. CTDA and CDTC are responsible for delivering the active subunit CDTB, which enters the host cell nucleus as a deoxyribonuclease, leading to cell cycle arrest and death. The CDTB protein selected in this invention shares over 90% homology with 36 other species of the Campylobacter genus and can also serve as a target protein for recognizing Campylobacter.

[0044] 2. CadF antibody and CdtB antibody selected by specific target protein CadF and CDTB screening are coupled to magnetic beads and mixed to obtain mixed magnetic beads. The two types of magnetic beads form a synergistic effect.

[0045] The beneficial effects of this invention are:

[0046] 1. This invention fuses two specific target protein gene fragments of Campylobacter genus, and then uses Escherichia coli for recombinant expression. The resulting fusion protein can screen for antibodies targeting two different targets in a single cell fusion. The antibodies are then conjugated to magnetic beads. The screened antibodies can be used to enrich Campylobacter in feces, for species identification and sequencing, and for qualitative and quantitative detection of Campylobacter genus.

[0047] 2. By coupling antibodies that recognize microbial surface proteins to magnetic beads, and then collecting them through the magnetic field of a magnetic rack, followed by antigen-antibody dissociation, the target microorganisms can be obtained. This method yields highly efficient and specific microorganisms, and can also be used for the isolation and screening of difficult-to-culture microorganisms.

[0048] 3. The method for enriching Campylobacter spp. in this invention is simple, does not require expensive instruments such as flow cytometers, reduces costs, and is conducive to widespread application. Attached Figure Description

[0049] Figure 1 Electrophoresis images of two monoclonal antibodies. Detailed Implementation

[0050] The present invention will now be described in further detail with reference to specific embodiments, so that those skilled in the art can understand it.

[0051] Example 1

[0052] Protein recombinant expression, purity and concentration detection

[0053] The full-length amino acid sequence of Campylobacter fibronectin-binding outer membrane protein CadF (Uniprot: A0A6F9MR14) is shown in SEQ ID NO: 4, and the codon-optimized nucleotide sequence is shown in SEQ ID NO: 3; the full-length amino acid sequence of Campylobacter cytotoxic protein CDTB (Uniprot: A4F283) is shown in SEQ ID NO: 6, and the codon-optimized nucleotide sequence is shown in SEQ ID NO: 5.

[0054] The codon-optimized nucleotide sequences of proteins CadF and CDTB were fused together. The amino acid sequence of the fused protein CadF-CDTB is shown in SEQ ID NO: 2, and the nucleotide sequence is shown in SEQ ID NO: 1.

[0055] The genes CadF-CDTB, CadF, and CDTB were ligated into the *E. coli* pET28a vector and transformed into *E. coli* BL21. The proteins CadF-CDTB, CadF, and CDTB were expressed in *E. coli*. After sonication and centrifugation, the supernatant was purified using a nickel column. The A280 concentration of the proteins was measured using a micro spectrophotometer, and the protein purity was analyzed by SDS-PAGE. The results are shown in Table 1. The purity of the purified proteins CadF-CDTB, CadF, and CDTB reached over 90%, indicating high purity.

[0056] Table 1. Protein concentration and purity results after purification.

[0057] protein name Purification Tag A280 concentration SDS-PAGE protein purity CadF-CDTB His 1.8 mg / mL 93％ CadF His 1.4 mg / mL 92％ CDTB His 1.3 mg / mL 90％

[0058] Example 2

[0059] Mouse immune and antiserum titers and hybridoma cell fusion

[0060] 1. Mouse immunization

[0061] (1) After mixing the protein CadF-CDTB with Freund's complete adjuvant, emulsify it in a mixer. The initial immunization dose is 50 μg of protein, and the second to fourth immunizations are also 50 μg of protein, immunized every two weeks. After 3 to 4 immunizations, take tail vein blood from mice and detect the serum antibody titer. Mice with a titer of 1:10,000 or higher are generally selected for fusion.

[0062] (2) Three days prior to fusion, mice were immunized with a shock dose of 100 μg protein per mouse, without adjuvant. The immunization method is shown in Table 2:

[0063] Table 2. Mouse Immunization Schedule

[0064] Number of immunizations Immune sites adjuvant Immunization dose 1 subcutaneous Freund's complete adjuvant 50μg 2 abdominal cavity Freund's incomplete adjuvant 50μg 3 abdominal cavity Freund's incomplete adjuvant 50μg 4 abdominal cavity Freund's incomplete adjuvant 50μg 5 abdominal cavity No adjuvants 100μg

[0065] 2. Hybridoma cell fusion

[0066] (1) After blood was collected from the eyeballs of mice after immunization, spleen cell suspension was prepared from mice with good immunization effect and washed with PBS. SP2 / 0 cells were then mixed in at a ratio of spleen cells:SP2 / 0 = 10:1. After centrifugation at 1000 rpm for 5 min, the mixed cells were drained and the cell clumps were loosened by tapping.

[0067] (2) Add 1 mL of PEG1450 to a 37°C water bath. After adding the PEG1450, react in a 37°C water bath for 2 min. Then slowly add 20 mL of RPMI-1640 stop solution along the tube wall.

[0068] (3) After the cell fusion was terminated, the cells were centrifuged at 800 rpm for 5 min and the residual liquid was aspirated. The cells were resuspended in DMEM complete medium containing HAT and pipetted into 96-well cell culture plates using a multichannel pipette.

[0069] (4) Cell colonies can generally be observed 3 days after fusion, and the medium should be changed and tested after 7 days.

[0070] (5) Based on cell growth, when the colony size reaches approximately 1 / 4 of the bottom area of ​​the well, detection can be considered. Take 100 μL of supernatant and perform detection using the indirect ELISA method. Select positive wells with high OD values ​​and good colony status for subcloning.

[0071] (6) Using HT medium, the selected positive cell colonies were diluted to 1 cell / well using the limiting dilution method. The cells were then seeded into 96-well cell culture plates. Once the monoclonal cells reached a medium size and a density of approximately 102, the cells were allowed to grow. 4 The titer can be detected with a number of cells or more. Then, positive cell wells are taken again for subclonal screening. Once all cell supernatants in the microwells are positive, the same subcloning process is repeated until a completely positive result is obtained again, confirming the selection of a positive hybridoma cell line. In this example, two cell lines were selected, numbered 1A10 and 3D9, respectively. These were coated with CadF-CDTB, CadF, and CDTB proteins, targeting recognition sites C65 and A61. The results are shown in Table 3. The results indicate that the two antibodies recognize CadF and CDTB proteins, respectively. This example successfully obtained two monoclonal antibodies, monoclonal antibody 1A10 and monoclonal antibody 3D9.

[0072] Table 3. Cell line recognition site detection

[0073] protein name Antibody 1A10 3D9 antibody CadF-CDTB + + CadF + - CDTB - +

[0074] Note: "+" indicates that the antigen and antibody react, and "-" indicates that the antigen and antibody do not react.

[0075] 3. Antiserum titer detection

[0076] The titers of the two antibodies were detected by indirect ELISA, as shown in Table 4. The monoclonal antibodies were identified using an antibody subtype identification kit, and the subtype results are shown in Table 5. The subtype of monoclonal antibody 1A10 was IgG2a, and the subtype of monoclonal antibody 3D9 was IgG1.

[0077] Table 4. Titer determination of the two monoclonal antibodies

[0078] Antibody dilution factor (starting dilution at 1 mg / ml) 1A10 antibody OD450 3D9 antibody OD450 1∶5000 3.98 3.97 1∶10000 3.67 3.65 1∶20000 3.24 2.98 1∶40000 2.98 2.65 1∶80000 2.54 2.12 1∶160000 1.98 1.86 1∶320000 1.56 1.47 1∶640000 1.22 1.13 1∶1280000 0.96 0.77 1∶2560000 0.64 0.46 0 (Negative control) 0.03 0.04

[0079] Table 5. Isotype determination of the two monoclonal antibodies

[0080] Monoclonal antibody number Subtype 1A10 IgG2a 3D9 IgG1

[0081] Example 3

[0082] Preparation and purification of ascites fluid for monoclonal antibodies

[0083] 1. One week before inoculation with hybridoma cells, BALB / c mice were intraperitoneally injected with 0.5 mL of Freund's incomplete adjuvant per mouse, followed by inoculation with 5 × 10⁶ cells per mouse. 6 A number of positive hybridoma cells were collected, and ascites fluid was collected 7–12 days later to determine the monoclonal antibody titer.

[0084] 2. Centrifuge the collected ascites fluid at 10,000 rpm for 10 min to remove cellular components and other precipitates. Collect the supernatant and test the monoclonal antibody titer. Aliquot and store at -80℃ for later use.

[0085] 3. Load the monoclonal antibody sample to be purified onto a Protein A-agarose affinity chromatography column at a flow rate of 0.5 mL / min to allow the antibody to bind to Protein A. Finally, elute with elution buffer to obtain monoclonal antibody 1A10 and monoclonal antibody 3D9. Identify their purity using SDS-PAGE. Figure 1 As shown in Table 6, the concentrations of the two antibodies were determined using the NanoDrop method. Monoclonal antibodies 1A10 and 3D9 exhibited high purity.

[0086] Table 6. Concentrations of the two monoclonal antibodies

[0087] Antibody name A280 concentration 1A10 5.2 mg / mL 3D9 4.8 mg / mL

[0088] Example 4

[0089] Cell line sequencing

[0090] 1. Hybridoma cells 1A10 and 3D9 were cultured and lysed. Total RNA and mRNA were extracted from the lysates. cDNA was synthesized by reverse transcription of the mRNA using random hexamer primers (5'-Pd(NNNNNN)-3'N = G, A, T, or C). Then, two rounds of nested PCR were performed: amplification was performed using the first-strand cDNA as a template. The forward primer was a sequence complementary to the corresponding heavy and light chain leader sequences, and the reverse primer was a sequence within the constant regions of the heavy and light chains. The forward and reverse primers were identical for hybridoma cells 1A10 and 3D9.

[0091] Heavy chain forward primer: CGGCCCAGCCGGCC;

[0092] Heavy chain reverse primer: TGAACCGCCTCCACC;

[0093] Light chain forward primer: GGTTCCACTGGT;

[0094] Light chain reverse primer: GTGCAGCATCAGC.

[0095] The PCR amplification program was as follows: denaturation at 94℃ for 2 min; denaturation at 94℃ for 20 s, annealing at 58℃ for 20 s, extension at 72℃ for 60 s, for 40 PCR cycles; final extension at 72℃ for 5 min.

[0096] 2. The second round of amplification yielded gene products with restriction enzyme sites (EcoRI and HindIII), which were ligated into the cloning vector pMD19-T. Then, through sequencing and analysis, the light chain and heavy chain variable region sequences of monoclonal antibody 1A10 and monoclonal antibody 3D9 were obtained.

[0097] 3. Light chain and heavy chain variable region sequences of monoclonal antibody 1A10 and monoclonal antibody 3D9 (1) The amino acid sequence of the light chain variable region of monoclonal antibody 1A10 is shown in SEQ ID NO: 7:

[0098]

[0099] Note: The bold and underlined regions indicate the complementarity-determining region (1A10-CDR-L) of the monoclonal antibody 1A10, while the region marked only in bold is the light chain backbone region (1A10-FR-L). The amino acid sequences of 1A10-CDR-L and 1A10-FR-L are as follows:

[0100] 1A10-FR-L1:DIVLTQSPASLAVSLGQRATISY;

[0101] 1A10-CDR-L1:HGWRWPDCWACWNFR;

[0102] 1A10-FR-L2:WNQQKPGQPPRLLIY;

[0103] 1A10-CDR-L2: APWCADW;

[0104] 1A10-FR-L3: GVPARFSGSGSGTDFTLNIHPVEEEDAATYYC;

[0105] 1A10-CDR-L3: SRVH;

[0106] 1A10-FR-L4:FGGGTKLEIK.

[0107] (2) The amino acid sequence of the variable region of the heavy chain of monoclonal antibody 1A10 is shown in SEQ ID NO: 8:

[0108]

[0109] Note: The bold and underlined regions indicate the complementarity-determining region (1A10-CDR-H) of the monoclonal antibody 1A10 heavy chain, while the region marked only in bold is the heavy chain backbone region (1A10-FR-H). The amino acid sequences of 1A10-CDR-H and 1A10-FR-H are as follows:

[0110] 1A10-FR-H1:EVLLQQSGPELVKPGASVKISCKASGYAFT;

[0111] 1A10-CDR-H1: ENCPE;

[0112] 1A10-FR-H2:WVKQSHGKSLEWIG;

[0113] 1A10-CDR-H2:EVCFCCAACVNCSRPHA;

[0114] 1A10-FR-H3:KATLTVDKSSSTAYMEVRSLTSEDTAVYYCAR;

[0115] 1A10-CDR-H3: KGLWFASAPENS;

[0116] 1A10-FR-H4:WGQGTSVTVSS.

[0117] (3) The amino acid sequence of the variable region of the light chain of monoclonal antibody 3D9 is shown in SEQ ID NO: 9:

[0118]

[0119] Note: The bold and underlined regions indicate the complementarity-determining region (3D9-CDR-L) of the 3D9 light chain, while the region marked only in bold is the 3D9 light chain backbone region (3D9-FR-L). The amino acid sequences of 3D9-CDR-L and 3D9-FR-L are as follows:

[0120] 3D9-FR-L1:DVVMTQTPLSLPVSLGDQASISC;

[0121] 3D9-CDR-L1: HWWSTAIRWCACQPAR;

[0122] 3D9-FR-L2:WYLQKPGQSPKLLIY;

[0123] 3D9-CDR-L2: RPWCHPW;

[0124] 3D9-FR-L3: GVPDRFSGSGSGTDFTLKISRVEAEDLGVYFC;

[0125] 3D9-CDR-L3: WTWCKPKCN;

[0126] 3D9-FR-L4:FGGGTKLEIK.

[0127] (4) The amino acid sequence of the variable region of the heavy chain of monoclonal antibody 3D9 is shown in SEQ ID NO: 10:

[0128]

[0129] Note: The bold and underlined regions indicate the complementarity-determining region (3D9-CDR-H) of the 3D9 heavy chain of the monoclonal antibody, while the region marked only in bold is the 3D9 heavy chain backbone region (3D9-FR-H). The amino acid sequences of 3D9-CDR-H and 3D9-FR-H are as follows:

[0130] 3D9-FR-H1: LEVVKPGTSVKLSCTASGFNFN;

[0131] 3D9-CDR-H1: SCNFW;

[0132] 3D9-FR-H2: WVKQRPEQGLEWIG;

[0133] 3D9-CDR-H2:KVEFGVAENSWEFRPRW;

[0134] 3D9-FR-H3:KATITADKSSNTAYLQLSSLTSEDSAVYYCAR;

[0135] 3D9-CDR-H3: PFANSQT;

[0136] 3D9-FR-H4:WGLGTTLAVSS.

[0137] Example 5

[0138] Preparation of antibody-conjugated magnetic beads

[0139] 1. Dilute the monoclonal antibody

[0140] The buffers for monoclonal antibody 1A10 and monoclonal antibody 3D9 were replaced with 15mM MES buffer (pH 6.0), and the antibodies were diluted to 2mg / mL with MES buffer to obtain diluted monoclonal antibody 1A10 and monoclonal antibody 3D92.

[0141] 2. Activation of carboxyl groups on the surface of magnetic beads

[0142] (1) After mixing the magnetic beads, take 100 μL of Mag COOH magnetic beads into a 1 mL centrifuge tube, remove the supernatant by magnetic separation, wash twice with 200 μL of MEST solution (100 mM MES, pH 5.0, 0.05% Tween 20), and then remove the supernatant.

[0143] (2) Quickly add 100 μL of freshly prepared EDC solution (10 mg / mL, using the above MEST solution as a dispersant) and 100 μL of NHS solution (10 mg / mL, using the above MEST solution as a dispersant) to the centrifuge tube containing the magnetic beads, vortex to mix and fully suspend the magnetic beads, activate at 25°C for 30 min, during which time keep the magnetic beads in suspension (a vertical mixer can be used for inverted mixing).

[0144] After the above steps, the carboxyl groups on the surface of the magnetic beads have been activated, resulting in an activated carboxyl magnetic bead solution, which can be covalently coupled with biological ligands containing primary amino groups (the activated state should not be stored for a long time; it is recommended to perform coupling immediately).

[0145] 3. Covalent conjugation of magnetic beads with monoclonal antibodies 1A10 and 3D9

[0146] (1) Take 200 μg of diluted monoclonal antibody 1A10 and monoclonal antibody 3D9 and mix them with 100 μL (10 μm in diameter) of the above activated carboxyl magnetic bead solution. After coupling at 25°C for 1 h, place at 4°C overnight. Keep the magnetic beads in suspension during coupling (you can use a vertical mixer to invert and mix them).

[0147] (3) Magnetic separation: aspirate the supernatant and simultaneously detect the remaining antibody content in the supernatant. Calculate the amount and concentration of the magnetic bead-conjugated antibody. Wash the magnetic beads 2 to 3 times with physiological saline and resuspend them with physiological saline to obtain antibody-conjugated magnetic beads 1A10 and 3D9, namely 1A10 magnetic beads and 3D9 magnetic beads.

[0148] Example 6

[0149] Enrichment of Campylobacter

[0150] (4) Mix 1A10 magnetic beads and 3D9 magnetic beads at a magnetic bead mass ratio of 1:1 to obtain mixed antibody-conjugated magnetic beads.

[0151] (1) Take 5 mL of fecal suspension containing Campylobacter (5 g of feces was suspended in 5 mL of physiological saline), add 0.1 mg of mixed antibody-conjugated magnetic beads, mix and incubate at 37 °C for 2 h, and use 1A10 magnetic beads and 3D9 magnetic beads added separately as controls.

[0152] (2) The mixed antibody-conjugated magnetic beads were separated using a magnetic rack, and the microorganisms and supernatant that were not bound to the mixed antibody-conjugated magnetic beads were removed. Then, the mixed antibody-conjugated magnetic beads (i.e., labeled magnetic beads) bound to Campylobacter were resuspended in physiological saline. The labeled magnetic beads were then mixed with an antibody label removal reagent, i.e., 0.05% papain, and incubated at 37°C for 0.5 h to cleave the Fc and Fab of the mouse monoclonal antibody, thereby separating the mixed antibody-conjugated magnetic beads from Campylobacter.

[0153] (3) Then, the mixed antibody-conjugated magnetic beads were collected using a magnetic rack, and the supernatant was the Campylobacter suspension. After diluting the Campylobacter, it was added to a hemocytometer and counted under a microscope. The yield was used to calculate the enrichment effect of using a 1:1 mass ratio of 1A10 and 3D9 magnetic beads for Campylobacter enrichment. The results are shown in Table 7. The effect of the mixed antibody-conjugated magnetic beads was greater than that of the single magnetic beads.

[0154] Table 7. Types and yields of antibody-conjugated magnetic beads combinations

[0155]

[0156]

[0157] Example 7

[0158] Campylobacter culture and sequencing

[0159] (1) Dilute the Campylobacter isolated in Example 6 to 10 3 ~10 4 The cells were collected at a concentration of 100 cells / mL and then spread onto blood agar medium. The culture was then placed in a 42°C incubator for anaerobic incubation for 48 hours. The colony morphology was observed: the bacteria were spiral or curved rod-shaped, with a size of (0.2-0.8) μm × (0.5-5) μm, and could be up to 8 μm long. They had diverse shapes, including S-shaped or seagull-like shapes.

[0160] (2) Pick 50 single colonies and use them as templates for PCR amplification of 16S rDNA.

[0161] Forward primer: 5'-AGAGTTTGATCMTGGCTCAG-3';

[0162] Reverse primer: 5'-GGTTACGTTACGACTT-3'.

[0163] PCR reaction system: DNA template (10 ng / μL) 1 μL, forward and reverse primers (10 μmol / L) 2 μL each, 10×PCR Buffer 5 μL, dNTPs (2.5 mmol / L) 4 μL, Taq enzyme (5 U / μL) 0.5 μL, ddH2O 35.5 μL. PCR reaction conditions: 94℃ for 3 min; 94℃ for 30 s, 56℃ for 1 min, 72℃ for 90 s, 72℃ for 10 min, 30 cycles.

[0164] (3) After separation by agarose gel electrophoresis, a specific band of about 1500 bp was visible. The target band was purified by gel recovery and nucleotide sequencing. The sequencing results were compared with the NCBI database by BLAST. The results showed that the 16S rDNA gene sequences of 50 colonies were compared with Campylobacter. Among them, 30 were Campylobacter jejuni, 10 were Campylobacter coli, 5 were Campylobacter fetus, 3 were Campylobacter guinea, and 2 were Campylobacter embryonic.

[0165] All other parts not described in detail are existing technologies. Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A monoclonal antibody against a Campylobacter genus-specific protein, characterized in that: The monoclonal antibodies include monoclonal antibody 1A10 and monoclonal antibody 3D9; Monoclonal antibody 1A10 includes a light chain variable region and a heavy chain variable region. The amino acid sequence of the light chain variable region of monoclonal antibody 1A10 is shown in SEQ ID NO: 7, and the amino acid sequence of the heavy chain variable region of monoclonal antibody 1A10 is shown in SEQ ID NO:

8. Monoclonal antibody 3D9 includes a light chain variable region and a heavy chain variable region. The amino acid sequence of the light chain variable region of monoclonal antibody 3D9 is shown in SEQ ID NO: 9, and the amino acid sequence of the heavy chain variable region of monoclonal antibody 3D9 is shown in SEQ ID NO:

10.

2. The monoclonal antibody according to claim 1, characterized in that: The light chain variable region of the monoclonal antibody 1A10 includes three complementarity-determining regions, namely: 1A10-CDR-L1:HGWRWPDCWACWNFR; 1A10-CDR-L2: APWCADW; 1A10-CDR-L3: SRVH; The heavy chain variable region of monoclonal antibody 1A10 includes three complementarity-determining regions, namely: 1A10-CDR-H1: ENCPE; 1A10-CDR-H2:EVCFCCAACVNCSRPHA; 1A10-CDR-H3: KGLWFASAPENS; The light chain variable region of monoclonal antibody 3D9 includes three complementarity-determining regions, namely: 3D9-CDR-L1: HWWSTAIRWCACQPAR; 3D9-CDR-L2: RPWCHPW; 3D9-CDR-L3: WTWCKPKCN; The heavy chain variable region of monoclonal antibody 3D9 includes three complementarity-determining regions, namely: 3D9-CDR-H1: SCNFW; 3D9-CDR-H2: KVEFGVAENSWEFRPRW; 3D9-CDR-H3: PFANSQT.

3. The monoclonal antibody according to claim 1, characterized in that: The monoclonal antibody 1A10 and monoclonal antibody 3D9 were prepared from hybridoma cell lines.

4. A method for preparing a hybridoma cell line, characterized in that: Includes the following steps: (1) The nucleotide sequences of the codon-optimized proteins CadF and CDTB were fused to obtain the fusion protein CadF-CDTB; (2) The nucleotide sequence of the fusion protein CadF-CDTB was transformed into Escherichia coli BL21, expressed, and purified by sonication to obtain the purified protein; (3) Mix the purified protein with Freund's adjuvant, emulsify it and then immunize the mice; (4) Then, spleen cells from immunized mice were fused with myeloma cells SP2 / 0, and hybridoma cell lines 1A10 and 3D9 were obtained through screening.

5. The preparation method according to claim 4, characterized in that: The amino acid sequence of the protein CadF is shown in SEQ ID NO: 4, the amino acid sequence of the protein CDTB is shown in SEQ ID NO: 6, and the amino acid sequence of the fusion protein CadF-CDTB is shown in SEQ ID NO:

2.

6. The preparation method according to claim 4, characterized in that: The optimized nucleotide sequence of the protein CadF is shown in SEQ ID NO: 3, the optimized nucleotide sequence of the protein CDTB is shown in SEQ ID NO: 5, and the nucleotide sequence of the fusion protein CadF-CDTB is shown in SEQ ID NO:

1.

7. The use of the monoclonal antibody as described in claim 1 in the preparation of antibody-conjugated magnetic beads.

8. A method for preparing antibody-conjugated magnetic beads, characterized in that: Includes the following steps: (1) Dilute monoclonal antibody 1A10 and monoclonal antibody 3D9 to 1.5-2.5 mg / mL using morpholine ethanesulfonic acid buffer to obtain diluted monoclonal antibody 1A10 and monoclonal antibody 3D9; (2) The magnetic beads were activated by carboxyl groups to obtain an activated carboxyl magnetic bead solution; (3) The diluted monoclonal antibody 1A10 and monoclonal antibody 3D9 were coupled with the activated carboxyl magnetic bead solution to obtain antibody-coupled magnetic beads 1A10 and antibody-coupled magnetic beads 3D9, wherein the particle size of the activated carboxyl magnetic beads was 10-30 μm, and the molar ratio of monoclonal antibody 1A10 or monoclonal antibody 3D9 to the activated carboxyl magnetic bead solution was 1:5-10.

9. The preparation method according to claim 8, characterized in that: The monoclonal antibody 1A10 and monoclonal antibody 3D9 were diluted to 2 mg / mL; The activated carboxyl magnetic beads have a particle size of 10 μm, and the molar ratio of monoclonal antibody 1A10 or monoclonal antibody 3D9 to the activated carboxyl magnetic bead solution is 1:

5.

10. The application of antibody-conjugated magnetic beads prepared by the method described in claim 8 in the enrichment of Campylobacter spp. bacteria, characterized in that: The antibody-conjugated magnetic beads are one or both of antibody-conjugated magnetic beads 1A10 and antibody-conjugated magnetic beads 3D9.

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