Antibodies against Clostridium difficile-specific proteins, magnetic bead-antibody conjugates and their applications

Abstract

The present invention discloses an antibody against Clostridium difficile specific protein, a magnetic bead-antibody conjugate and its application. The monoclonal antibody is any one of monoclonal antibody CD1, monoclonal antibody CD2, monoclonal antibody CD3 and monoclonal antibody CD4. The magnetic bead-antibody conjugate is formed by conjugating a monoclonal antibody with activated carboxyl magnetic beads to obtain the magnetic bead-antibody conjugate. Based on the magnetic bead-antibody conjugate sorting method, antibody magnetic beads that couple and recognize the Clostridium difficile secreted proteins TcdA, TcdB, the highly expressed GDH on the surface of Clostridium difficile, and the LPxTG of Clostridium difficile are used to capture Clostridium difficile, achieving the purpose of rapidly isolating Clostridium difficile from fecal samples. The present invention is based on the immunomagnetic bead separation technology of antigen-antibody reaction for separation, with the characteristics of being rapid, simple, having high enrichment efficiency and strong specificity. The present invention can be used to isolate and remove Clostridium difficile in fecal microbiota transplantation, and can also be used for strain identification and sequencing.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to an antibody against a Clostridium difficile-specific protein, a magnetic bead-antibody conjugate and applications thereof. Background Art

[0002] Clostridium difficile (CD), originally named Bacillus difficile, is a Gram-positive, flagellated, obligately anaerobic bacillus. It is primarily transmitted via the fecal-oral route as spores and is widely distributed in the intestines of humans and animals, as well as in the environment. CD has a strong survival ability, with its spores able to survive for months in the natural environment, making it difficult to kill with ordinary disinfectants. C. difficile is an anaerobic bacterium, which grows better in the absence of oxygen than in aerobic environments. The human intestine happens to be a relatively oxygen-free environment.

[0003] Under normal circumstances, the human intestinal microbial flora maintains a well-balanced homeostasis. However, long-term or irregular use of broad-spectrum antibiotics, immunosuppressants, hormones, and chemotherapeutic agents, as well as ageing, can weaken intestinal colonization, disrupt intestinal flora, and allow Clostridium difficile (CD) bacteria to proliferate, releasing toxins that damage intestinal mucosal cells and the intestinal wall, leading to Clostridium difficile infection (CDI), characterized by fever, abdominal pain, and diarrhea. Clinical manifestations can range from mild, self-limited diarrhea to severe pseudomembranous colitis and even toxic megacolon, which can lead to intestinal perforation, intra-abdominal infection, and sepsis.

[0004] Currently, testing for C. difficile mainly involves strain detection and C. difficile toxin detection. In cases of healthcare-associated diarrhea or any case of unexplained diarrhea, C. difficile and its toxins should be systematically tested. Two-step diagnostic methods offer high sensitivity and specificity and are therefore more suitable for the diagnosis of CDI. Detection of C. difficile in stool samples can be affected by a variety of other bacteria and components, necessitating sample pretreatment to remove interfering substances and significant background signal interference, thereby improving the sensitivity and specificity of subsequent detection methods. Furthermore, prior to testing, the target bacteria in the sample must be concentrated and enriched to achieve the detection limit of the assay. Bacterial enrichment techniques can rapidly concentrate the target bacteria in a sample within tens of minutes. Among the many enrichment techniques used in sample processing, immunomagnetic separation based on antigen-antibody reactions is currently a hot topic. Antibodies that recognize microbial surface proteins are coupled to magnetic beads, which are then separated and collected using a magnetic field. The target microorganisms are then separated through antigen-antibody dissociation. This method is highly efficient and specific in identifying microorganisms and can be used to isolate and screen difficult-to-cultivate microorganisms. Summary of the Invention

[0005] The purpose of the present invention is to overcome the shortcomings of the existing technology and provide an antibody, magnetic bead antibody conjugate and its application to a Clostridium difficile-specific protein. The present invention selects Clostridium difficile secretory proteins TcdA and TcdB and GDH, which is highly expressed on the surface of Clostridium difficile, and Clostridium difficile LPxTG as target proteins. Using the above four proteins, Balb / C mice are immunized, and monoclonal antibodies are prepared by hybridoma technology. The monoclonal antibodies are then conjugated to magnetic beads. Based on the antibody conjugate sorting method, antibody magnetic beads that are conjugated to recognize Clostridium difficile secretory proteins TcdA and TcdB and GDH, which is highly expressed on the surface of Clostridium difficile, and Clostridium difficile LPxTG are used to capture Clostridium difficile, thereby achieving the purpose of rapid and specific isolation of Clostridium difficile from fecal samples.

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

[0007] The present invention provides a monoclonal antibody against a target protein of Clostridium difficile, wherein the monoclonal antibody is any one of monoclonal antibody CD1, monoclonal antibody CD2, monoclonal antibody CD3 and monoclonal antibody CD4, wherein:

[0008] Monoclonal antibody CD1 includes CD1 heavy chain and CD1 light chain, and their amino acid sequences are:

[0009] The amino acid sequence of the CD1 heavy chain is shown in SEQ ID NO: 9:

[0010]

[0011] The amino acid sequence of the CD1 light chain is shown in SEQ ID NO: 10:

[0012]

[0013] Monoclonal antibody CD2 includes CD2 heavy chain and CD2 light chain, and their amino acid sequences are:

[0014] The amino acid sequence of the CD2 heavy chain is shown in SEQ ID NO: 11:

[0015]

[0016] The amino acid sequence of the CD2 light chain is shown in SEQ ID NO: 12:

[0017]

[0018] Monoclonal antibody CD3 includes CD3 heavy chain and CD3 light chain, and their amino acid sequences are:

[0019] The amino acid sequence of the CD3 heavy chain is shown in SEQ ID NO: 13:

[0020]

[0021] The amino acid sequence of the CD3 light chain is shown in SEQ ID NO: 14:

[0022]

[0023] Monoclonal antibody CD4 includes CD4 heavy chain and CD4 light chain, and their amino acid sequences are:

[0024] The amino acid sequence of the CD4 heavy chain is shown in SEQ ID NO: 15:

[0025]

[0026] The amino acid sequence of the CD4 light chain is shown in SEQ ID NO: 16:

[0027]

[0028] Furthermore, in the monoclonal antibody CD1, the CD1 heavy chain amino acid sequence includes three complementary determining regions (CDRs), namely:

[0029] CD1-CDR-H1: RWYKD,

[0030] CD1-CDR-H2: IEPNSDGYNITYSPKLD,

[0031] CD1-CDR-H3:LDYTSNSN;

[0032] The amino acid sequence of the CD1 light chain includes three complementarity determining regions (CDRs), namely:

[0033] CD1-CDR-L1: WHLSQINSHLG,

[0034] CD1-CDR-L2: VVSKLDSG,

[0035] CD1-CDR-L3:WQGWT;

[0036] In the monoclonal antibody CD2, the CD2 heavy chain amino acid sequence includes three complementary determining regions (CDRs), namely:

[0037] CD2-CDR-H1:SYWLH,

[0038] CD2-CDR-H2: YGISPASTAPGNSATDK,

[0039] CD2-CDR-H3:GNNY;

[0040] The CD2 light chain amino acid sequence includes three complementarity determining regions (CDRs):

[0041] CD2-CDR-L1: KSSQSLLDSDGKTYLN,

[0042] CD2-CDR-L2: VVSKLDS,

[0043] CD2-CDR-L3:WQGWT;

[0044] In the monoclonal antibody CD3, the CD3 heavy chain amino acid sequence includes three complementary determining regions (CDRs), namely:

[0045] CD3-CDR-H1:RNDGI

[0046] CD3-CDR-H2:HIRNDAEGIWMGQKDET

[0047] CD3-CDR-H3:TRMADCQMF

[0048] The CD3 light chain amino acid sequence includes three complementarity determining regions (CDRs):

[0049] CD3-CDR-L1: ADNCQGEQIKETSTWY,

[0050] CD3-CDR-L2: HIMGDET,

[0051] CD3-CDR-L3:ARTWGQDTE;

[0052] In the monoclonal antibody CD4, the CD4 heavy chain amino acid sequence includes three complementary determining regions (CDRs), namely:

[0053] CD4-CDR-H1:THQPFT,

[0054] CD4-CDR-H2:RADNCQTSMKFTPSWY,

[0055] CD4-CDR-H3:GHTMFTPSE;

[0056] The CD4 light chain amino acid sequence includes three complementarity determining regions (CDRs):

[0057] CD4-CDR-L1:RSALYYANENI,

[0058] CD4-CDR-L2:NLRHGST,

[0059] CD4-CDR-L3: TQDPKDWSL.

[0060] Furthermore, the monoclonal antibody is prepared from a hybridoma cell line.

[0061] The present invention also provides a method for preparing a hybridoma cell line, comprising the following steps:

[0062] 1) transforming the optimized nucleotide sequence of the protein into Escherichia coli BL21, expressing it, and ultrasonically purifying it to obtain a purified protein; wherein the protein is any one of protein TcdA, protein TcdB, protein GDH, and protein LPxTG;

[0063] 2) mixing the purified protein with Freund's adjuvant, emulsifying and immunizing mice; then taking spleen cells from the immunized mice and fusing them with myeloma cells SP2 / 0, and detecting and screening to obtain hybridoma cell lines, wherein the hybridoma cell line is any one of hybridoma cell line CD1, hybridoma cell line CD2, hybridoma cell line CD3 and hybridoma cell line CD4.

[0064] Furthermore, when the protein is protein TcdA, the optimized nucleotide sequence of protein TcdA is shown in SEQ ID NO: 2,

[0065] Alternatively, when the protein is protein TcdB, the optimized nucleotide sequence of protein TcdB is shown in SEQ ID NO: 4,

[0066] Alternatively, when the protein is GDH, the optimized nucleotide sequence of GDH is as shown in SEQ ID NO: 6.

[0067] Alternatively, when the protein is protein LPxTG, the optimized nucleotide sequence of protein LPxTG is shown in SEQ ID NO:8.

[0068] The basis for protein selection of the present invention:

[0069] In most CD strains, the pathogenicity locus typically encodes five proteins, located at a specific chromosomal location. The primary toxins encoded by the pathogenicity locus are TcdA (also known as ToxA) and TcdB (also known as ToxB), two secreted proteins. The toxins form a complex with specific receptors on the host cell surface and are internalized. Endosome acidification allows the C-terminal repetitive oligopeptide sequence (CROP) domain of the toxin protein to be embedded in the endosomal membrane, subsequently transporting the cysteine ​​protease domain (CPD) and glucosyltransferase domain (CTD) to the cytoplasm. Phytic acid (IP6) activates cysteine ​​proteases, cleaving and releasing the toxic glycotransferases. The CTD catalyzes the glycosylation of RHO or RAC CTPases, inactivating CTP-binding proteins, depolymerizing epithelial actin, and disrupting the cytoskeleton, ultimately leading to the destruction of intestinal tight junctions and epithelial integrity. Multiple studies have shown that while TedA and TcdB play a role in most infections, TedB is more important in determining disease severity. The locus also encodes three other proteins, TedR, TedC, and TedE. During steady-state growth, TcdR drives the transcription of TcdA and TcdB, promoting their protein expression. 2 During exponential growth, C. difficile expresses higher levels of TcdC, which inhibits the transcription of TcdA and TcdB, but the hypervirulence of C. difficile is associated with the loss of the TcdC sequence. TcdE encodes a choline-like protein that facilitates the secretion of TedA and TedB, which lack conventional secretion signal sequences.

[0070] Glutamate dehydrogenase (GDH) is a highly conserved metabolic enzyme and an antigenic protein. It is highly expressed and stable on the surface of all C. difficile bacteria. Enzyme immunoassays are commonly used to directly detect GDH in stool. GDH testing is low-cost, simple, rapid, and highly sensitive, making it a routine diagnostic tool for CDI. Because all C. difficile strains express high levels of GDH, GDH testing cannot distinguish between toxigenic and non-toxigenic strains. A positive GDH test only indicates the presence of C. difficile, while a negative GDH test excludes C. difficile infection. Therefore, GDH can be used as a biomarker for the presence of C. difficile in stool specimens, but it is not the preferred method for the rapid diagnosis of CDI.

[0071] Furthermore, the protein TcdA is shown as SEQ ID NO: 1, the protein TcdB is shown as SEQ ID NO: 3, the protein GDH is shown as SEQ ID NO: 5, and the protein LPxTG is shown as SEQ ID NO: 7.

[0072] The present invention also provides a use of the monoclonal antibody in preparing magnetic bead-antibody conjugates.

[0073] The present invention also provides a method for preparing a magnetic bead-antibody conjugate, comprising the following steps:

[0074] 1) Dilute the above monoclonal antibody using MES buffer,

[0075] 2) Activate the carboxyl group of the magnetic beads to obtain activated carboxyl magnetic beads

[0076] 3) coupling the monoclonal antibody of step 1) with activated carboxyl magnetic beads to obtain a magnetic bead antibody conjugate, wherein the magnetic bead antibody conjugate is any one of magnetic bead antibody conjugate CD1, magnetic bead antibody conjugate CD2, magnetic bead antibody conjugate CD3 and magnetic bead antibody conjugate CD4.

[0077] The particle size of the activated carboxyl magnetic beads is 10 to 30 μm, and the molar ratio of the monoclonal antibody to the activated carboxyl magnetic beads is 1:5 to 10.

[0078] Furthermore, the particle size of the activated carboxyl magnetic beads is 10 μm, and the molar ratio of the monoclonal antibody to the activated carboxyl magnetic beads is 1:5.

[0079] The present invention also provides an application of an antibody conjugate in enriching or separating Clostridium difficile, wherein the antibody conjugate is any one or more of magnetic bead antibody conjugate CD1, magnetic bead antibody conjugate CD2, magnetic bead antibody conjugate CD3 and magnetic bead antibody conjugate CD4.

[0080] Beneficial effects of the present invention:

[0081] 1. The present invention screened and obtained specific antibodies that recognize TcdA, TcdB, GDH, and LPxTG of Clostridium difficile. These four antibodies were coupled to magnetic beads. The four magnetic bead-antibody conjugates were used to enrich or isolate Clostridium difficile. These conjugates bind to different sites of the bacteria, creating a synergistic effect. This enrichment efficiency was improved by enriching the bacteria from the magnetic bead-coupled antibodies and multiple specific protein targets.

[0082] 2. The present invention does not require expensive instruments such as flow cytometry, and can be separated using a magnetic stand, which is convenient and relatively low-cost. This method is highly efficient and specific for the isolation of C. difficile. It can shorten the incubation time for C. difficile detection, remove substances in the matrix that affect the determination, and reduce the amount of sample required, thereby achieving rapid enrichment of C. difficile. Especially for samples with low levels, the enrichment technology is conducive to improving the accuracy of the analytical method and expanding the application range of the detection technology.

[0083] 3. The Clostridium difficile enriched or isolated from feces of the present invention can be used to separate and remove Clostridium difficile in microbiota transplantation, can also be used for identification and sequencing of bacterial species in CDI, and can also be used for the cultivation of the bacteria. BRIEF DESCRIPTION OF THE DRAWINGS

[0084] Figure 1 The electrophoresis diagram of the purified monoclonal antibodies from four hybridoma strains is shown in Figure 2. DETAILED DESCRIPTION

[0085] The present invention is further described in detail below with reference to specific embodiments so that those skilled in the art can understand.

[0086] Example 1 Protein recombinant expression purity and concentration detection

[0087] 1. Methods

[0088] Expression and purification of Clostridium difficile TcdA, TcdB, GDH and LPxTG proteins:

[0089] Escherichia coli was used to express the N-terminal 95-467aa of TcdA (UniProt accession number P16154), the N-terminal 96-469aa of TcdB (UniProt accession number Q9EXR0), the full-length 1-421aa of GDH (UniProt accession number P27346), and the full-length 1-220aa of LPxTG (UniProt accession number Q183R7), and the purity and concentration of the antigens were characterized.

[0090] 2. Conclusion

[0091] The codon-optimized nucleotide sequences of TcdA, TcdB, GDH, and LPxTG expressed in E. coli were transformed into E. coli BL21 and subjected to ultrasonic disruption. The supernatant after centrifugation was purified by nickel column to obtain the protein. The protein A280 concentration was measured by ultramicrospectrophotometer, and the protein purity was analyzed by SDS-PAGE (Table 1 below).

[0092] The amino acid sequence of protein TcdA is shown in SEQ ID NO: 1:

[0093] KNLHFVWIGGEVSDIALEYIKQWADINAEYNIKLWYDSEAFLVNTLKKAIVESSTTEALQLLEEEIQNPQFDNMKFYKKRMEFIYDRQKRFINYYKSQINKPTVPTIDDIIKSHLVSEYNRDETVLESYRTNSLRKINSNHGIDIRANSLFTEQELLNIYSQELLNRGNLAAASDIVRLLALKNFGGVYLDVDMLPGIHSDLFKTISRPSSIGLDRWEMIKLEAIMKYKKYINNYTSENFDKLDQQLKDNFKLIIESKSEKSEIFSKLENLNVSDLEIKIAFALGSVINQALISKQGSYLTNLVIEQVKNRYQFLNQHLNPAIESDNNFTDTTKIFHDSLFNSATAENSMFLTKIAPYLQVGFMPEARSTISL,

[0094] The nucleotide sequence of TcdA is shown in SEQ ID NO: 2:

[0095]

[0096] The amino acid sequence of protein TcdB is shown in SEQ ID NO: 3:

[0097] KNLHFIWIGGQINDTAINYINQWKDVNSDYNVNVFYDSNAFLINTLKKTIIESASNDTLESFRENLNDPEFNHTAFFRKRMQIIYDKQQNFINYYKAQKEENPDLIIDDIVKTYLSNEYSKDIDELNAYIEESLNKVTENSGNDVRNFEEFKTGEVFNLYEQELVERWNLAGASDILRVAILKNIGGVYLDVDMLPGIHPDLFKDINKPDSVKTAVDWEEMQLEAIMKYKEYIPEYTSKHFDTLDEEVQSSFESVLASKSDKSEIFLPLGGIEVSPLEVKVAFAKGSIIDQALISAKDSYCSDLLIKQIQNRYKILNDTLGPIISQGNDFNTTMNNFGESLGAIANEENISFIAKIGSYLRVGFYPEANTTITL,

[0098] The nucleotide sequence of TcdB is shown in SEQ ID NO: 4:

[0099]

[0100] The amino acid sequence of protein LPxTG is shown in SEQ ID NO:5:

[0101] DSTTIQQNKDTLSQIVVFPTGNYDKNEANAMVNRLANIDGKYLNALKQNNLKIKLLSGKLTDEKEYAYLKGVVPKGWEGTGKTWDDVPGLGGSTVALRIGFSNKGKGHDAINLELHETAHAIDHIVLNDISKSAQFKQIFAKEGRSLGNVNYLGVYPEEFFAESFAYYYLNQDTNSKLKSACPQTYSFLQNLAK,

[0102] The nucleotide sequence of LPxTG is shown in SEQ ID NO:6:

[0103] gacagcaccaccatccaacaaaacaaagataccctgtcccagattgttgtgttcccgacgggtaactacgataaaaacgaggccaacgcaatggttaaccgtctggcaaacatcgacggtaagtacctgaacgcgctgaagcagaacaacctgaaaatcaaactgctgtccggtaaactgacggacgaaaaagagtacgcctatctgaaaggtgttgttccgaaaaggttgggaaggtactggcaaaacctgggacgatgtgccgggtctgggtggtagcaccgttgcactgcgtattggtttctccaacaaaggtaaaggccacgacgcaatcaacctggagctgcacgaaacggcccatgctatcgatcacatcgtgctgaacgacatttccaaatctgcgcagtttaaacagatcttcgcaaaggaaggtcgtagcctgggcaacgtcaactacctgggtgtttatccggaagaatttttcgccgagtctttcgcctactactacctgaaccaggatacgaactctaaactgaaaagcgcttgcccgcaaacttatagcttcctgcagaacctggcaaaa;

[0104] The amino acid sequence of protein GDH is shown in SEQ ID NO:7:

[0105] MSGKDVNVFEMAQSQVKNACDKLGMEPAVYELLKEPMRVIEVSIPVKMDDGSIKTFKGFRSQHNDAVGPTKGGIRFHQNVSRDEVKALSIWMTFKCSVTGIPYGGGKGGIIVDPSTLSQGELERLSRGYIDGIYKLIGEKVDVPAPDVNTNGQIMSWMVDEYNKLTGQSSIGVITGKPVEFGGSLGRTAATGFGVAVTAREAAAKLGIDMKKAKIAVQGIGNVGSYTVLNCEKLGGTVVAMAEWCKSEGSYAIYNENGLDGQAMLDYMKEHGNLLNFPGAKRISLEEFWASDVDIVIPAALENSITKEVAESIKAKLVCEAANGPTTPEADEVFAERGIVLTPDILTNAGGVTVSYFEWVQNLYGYYWSEEEVEQKEEIAMVKAFESIWKIKEEYNVTMREAAYMHSIKKVAEAMKLRGWY,

[0106] The GDH nucleotide sequence is as shown in SEQ ID NO:8:

[0107]

[0108] Table 1 Concentration and purity of the four proteins after purification

[0109] Protein name Purification tags A280 concentration Protein purity by SDS-PAGE TcdA His 1.8mg / ml 94％ TcdB His 1.4mg / ml 93％ LPxTG His 2.3mg / ml 92％ GDH His 1.2mg / ml 91％

[0110] Example 2 Mouse Immunization and Antiserum Titer Detection and Hybridoma Cell Fusion

[0111] Take protein TcdA, protein TcdB, protein LPxTG and protein GDH respectively, mix them with Freund's adjuvant, and place them in a mixer for emulsification. The first immunization dose is 50ug protein / mouse, the adjuvant is Freund's complete adjuvant, and the interval between the second immunization is 3 weeks; the second to fourth immunization doses are 50ug protein / mouse, the adjuvant is Freund's incomplete adjuvant, and the interval between the second immunization is 2 weeks. Take the tail vein blood of the mouse and test the serum antibody titer. Generally, mice with a titer of 1:10,000 or more are selected for fusion. The immunization method is shown in Table 2:

[0112] Table 2 Mouse immunization schedule

[0113] Number of immunizations immune site adjuvant Immunization dose 1 subcutaneous Freund's complete adjuvant 50ug 2 abdominal cavity Freund's incomplete adjuvant 50ug 3 abdominal cavity Freund's incomplete adjuvant 50ug 4 abdominal cavity Freund's incomplete adjuvant 50ug 5 abdominal cavity Without adjuvant 100ug

[0114] Three days before fusion, pulse immunize each mouse with 50 μg of protein without adjuvant. After eye bleeding, prepare a splenocyte suspension from a well-immunized mouse, wash with PBS, and then mix with SP2 / 0 cells in a ratio of 10:1 splenocytes:SP2 / 0 cells. Centrifuge at 1000 rpm for 5 minutes, then drip the mixture to remove any cell clumps. Add 1 ml of PEG-1450 to the tube in a 37°C water bath. Incubate in a 37°C water bath for 2 minutes, then slowly add 20 ml of RPMI-1640 stop solution along the tube wall. After fusion stops, centrifuge at 800 rpm for 5 minutes, and aspirate any remaining liquid. Resuspend the cells in complete DMEM medium containing HAT and transfer to a 96-well cell culture plate using a multichannel pipette. Colonies can generally be observed 3 days after fusion, and the medium should be changed for monitoring 7 days after fusion.

[0115] Based on cell growth, consider testing when colonies have grown to approximately 1 / 4 of the well bottom area. Take 100 μL of supernatant and test using an indirect ELISA. Select positive wells with high OD values ​​and good colony status for subcloning.

[0116] The positive cell colonies screened were diluted to 1 per well using HT medium by limiting dilution method, and the cells were plated in 96-well cell culture plates. When the monoclonal cells grew to a medium size and the density was about 10 4The titer can be tested if more than 10 cells are detected; then the positive cell wells are taken again and the subcloning screening is repeated. After the supernatants of the cells in all microwells are detected to be positive, the same subcloning is performed again until all positive results are obtained again. It can be determined that positive hybridoma cell lines have been screened at this time. The cell lines are numbered CD1, CD2, CD3 and CD4. The subtypes of the four monoclonal antibodies were identified using the antibody subtype identification kit. The subtype results are shown in Table 3:

[0117] Table 3 Isotype determination of four monoclonal antibodies

[0118] Cell line number Subtype CD1 IgG1 CD2 IgG2a CD3 IgG2b CD4 IgG1

[0119] Example 3 Serum-free culture of hybridoma cells and purification of antibodies

[0120] Resuscitate hybridoma cells in the presence of serum. During several consecutive cell passages, gradually reduce the serum ratio (e.g., from 20% serum to 15% serum to 10% serum and then to serum-free), and gradually increase the substitute additives to allow the hybridoma cells to adapt and continue to grow. When preparing monoclonal antibodies, resuscitate hybridoma cells in serum-free medium. When the cells are in good condition, increase the amount of medium and the container for culturing cells. When a large number of cells die, collect the liquid, centrifuge and take the supernatant, filter and collect, take the sample to be purified and load it on 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 eluent to obtain the antibody, and identify its purity by SDS-PAGE. Figure 1 shown.

[0121] Example 4 Cell Line Sequencing

[0122] Hybridoma cells CD1, hybridoma cells CD2, hybridoma cells CD3 and hybridoma cells CD4 were cultured and lysed, total RNA and mRNA were extracted from the lysates, and cDNA was synthesized by reverse transcription of mRNA using random hexamer primers (5'-Pd(NNNNNN)-3'N=G, A, T or C), followed by two rounds of nested PCR: the first-strand cDNA was used as a template for amplification, the forward primer had a sequence complementary to the corresponding heavy chain and light chain leader sequence, and the reverse primer was a sequence within the constant region of the heavy chain and light chain.

[0123] Heavy chain forward primer: CGGCCCAGCCGGCC;

[0124] Heavy chain reverse primer: TGAACCGCCTCCACC;

[0125] Light chain forward primer: GGTTCCACTGGT;

[0126] Light chain reverse primer: GTGCAGCATCAGC.

[0127] The PCR amplification program was as follows: denaturation at 94°C for 2 min; denaturation at 94°C for 20 s, annealing at 58°C for 20 s, extension at 72°C for 60 s, and 40 PCR cycles; and final extension at 72°C for 5 min.

[0128] The second round of amplification produced gene products with restriction enzyme sites, which were ligated into cloning vectors. Sequencing and analysis then yielded the light and heavy chain variable region sequences of monoclonal antibodies CD1, CD2, CD3, and CD4, respectively. (Variable regions contain regions where amino acids can undergo frequent changes and combinations, known as hypervariable regions (HVRs). Hypervariable regions are also known as complementarity-determining regions (CDRs). Hypervariable regions determine the idiotype of the antibody (anti-idiotypic antibody expression). The amino acid composition and arrangement of non-HVR sites in the variable region are relatively conservative, known as framework regions (FRs). Note: The CDRs are highlighted in bold and underlined.)

[0129] Monoclonal antibody CD1 includes CD1 heavy chain and CD1 light chain, and their amino acid sequences are:

[0130] The amino acid sequence of the CD1 heavy chain is shown in SEQ ID NO: 9:

[0131]

[0132] in,

[0133] CD1-FR-H1: GGGLVQPGGSLKLSCAASGFDFS,

[0134] CD1-CDR-H1: RWYKD,

[0135] CD1-FR-H2: WVRQAPGKGLEWIG,

[0136] CD1-CDR-H2: IEPNSDGYNITYSPKLD,

[0137] CD1-FR-H3:KFIISRDNAKHTLYLQMSKVRSEDTALYYCAR,

[0138] CD1-CDR-H3:LDYTSNSN,

[0139] CD1-FR-H4:WGQGTALTVSS;

[0140] The amino acid sequence of the CD1 light chain is shown in SEQ ID NO: 10:

[0141]

[0142] in,

[0143] CD1-FR-L1: DIVLTQSPATLSVTPGDSVSLSC,

[0144] CD1-CDR-L1: WHLSQINSHLG,

[0145] CD1-FR-L2: WYQQKSHESPRLLIK,

[0146] CD1-CDR-L2: VVSKLDSG,

[0147] CD1-FR-L3: GVPDRFYGSGSGTDFTLKISRVEAEDLGVYYC,

[0148] CD1-CDR-L3: WQGWT,

[0149] CD1-FR-L4: FGAGTKLELK;

[0150] The monoclonal antibody CD2 includes a CD2 heavy chain and a CD2 light chain, and their amino acid sequences are as follows: The amino acid sequence of the CD2 heavy chain is shown in SEQ ID NO: 11:

[0151]

[0152] in,

[0153] CD2-FR-H1: EVKLQESGTVLARPGTSVKMSCRASGYSFY,

[0154] CD2-CDR-H1:SYWLH,

[0155] CD2-FR-H2:WIKQRPGQGLEWVG,

[0156] CD2-CDR-H2: YGISPASTAPGNSATDK,

[0157] CD2-FR-H3: KATLTAVTSASTAYMELSSLTNEDSAVYYCIR,

[0158] CD2-CDR-H3:GNNY,

[0159] CD2-FR-H4: WGQGTTVTVSS;

[0160] The amino acid sequence of the CD2 light chain is shown in SEQ ID NO: 12:

[0161]

[0162] in,

[0163] CD2-FR-L1: DVVLTQSPLTLSVTIGQPASISC,

[0164] CD2-CDR-L1: KSSQSLLDSDGKTYLN,

[0165] CD2-FR-L2:WLFQRPGQSPKRLIY,

[0166] CD2-CDR-L2: VVSKLDS,

[0167] CD2-FR-L3: GVPDRFYGSGSGTDFTLKISRVEAEDLGVYYC,

[0168] CD2-CDR-L3: WQGWT,

[0169] CD2-FR-L4:FGGGTKLEI;

[0170] The monoclonal antibody CD3 includes a CD3 heavy chain and a CD3 light chain, and their amino acid sequences are as follows: The amino acid sequence of the CD3 heavy chain is shown in SEQ ID NO: 13:

[0171]

[0172]

[0173] in,

[0174] CD3-FR-H1:VKPGGSLKLSCAASGFTFS,

[0175] CD3-CDR-H1:RNDGI,

[0176] CD3-FR-H2:WVRQTPDKRLEWVA,

[0177] CD3-CDR-H2:HIRNDAEGIWMGQKDET,

[0178] CD3-FR-H3: RFTISRDQDKQTLYLQMSSLKSEDTAMYYCAS,

[0179] CD3-CDR-H3: TRMADCQMF,

[0180] CD3-FR-H4:WGQGTTVTVS;

[0181] The amino acid sequence of the CD3 light chain is shown in SEQ ID NO: 14:

[0182]

[0183] in,

[0184] CD3-FR-L1:DLVMTQTAPSVPPVTPGESVSISC,

[0185] CD3-CDR-L1: ADNCQGEQIKETSTWY,

[0186] CD3-FR-L2: WFLQRPGQSPQLLIY,

[0187] CD3-CDR-L2: HIMGDET,

[0188] CD3-FR-L3: GVPDRFSGSGSGTAFTLRISRVEAEDVGVYYC,

[0189] CD3-CDR-L3: ARTWGQDTE,

[0190] CD3-FR-L4:FGSGTKLEIK;

[0191] The monoclonal antibody CD4 includes a CD4 heavy chain and a CD4 light chain, and their amino acid sequences are as follows: The amino acid sequence of the CD4 heavy chain is shown in SEQ ID NO: 15:

[0192]

[0193] Among them, CD4-FR-H1:EVKLVESGPDLVKPSQSLSLTCTVTGYSIT, CD4-CDR-H1:THQPFT,

[0194] CD4-FR-H2:WVRQTPDKRLEWVA,

[0195] CD4-CDR-H2:RADNCQTSMKFTPSWY,

[0196] CD4-FR-H3:RISMTRDTSKNQFFLQLNSVTTEDTATYYCAR,

[0197] CD4-CDR-H3:GHTMFTPSE,

[0198] CD4-FR-H4:WGQGTSVTVSS;

[0199] The amino acid sequence of the CD4 light chain is shown in SEQ ID NO: 16:

[0200]

[0201] in,

[0202] CD4-FR-L1: DIVLTQSPASLSSASVGEIVTITC,

[0203] CD4-CDR-L1:RSALYYANENI,

[0204] CD4-FR-L2: WYQQKQGKSPQLLVY,

[0205] CD4-CDR-L2:NLRHGST,

[0206] CD4-FR-L3:GVPSRFSGSGSGTQFSLKINSLQPEDFGSYYC,

[0207] CD4-CDR-L3: TQDPKDWSL,

[0208] CD4-FR-L4: FGGDTKLEIK.

[0209] Example 5 Determination of Preparation Conditions of Magnetic Bead Antibody Conjugates

[0210] 1. Monoclonal Antibody Dilution

[0211] The monoclonal antibody CD1 was buffer exchanged into 15 mM MES buffer, pH 6.0, and the antibody was diluted to 2 mg / mL with MES buffer to obtain antibody diluent CD1;

[0212] 2. Activation of carboxyl groups on the surface of magnetic beads

[0213] After mixing the magnetic beads, transfer 100 μL of COOH magnetic beads (70113-5, Suzhou Beaver Biotechnology) of different particle sizes to a 1 mL centrifuge tube. Magnetic separation is performed to remove the supernatant, and the beads are magnetically separated and washed twice with 200 μL of MEST solution (100 mM MES, pH 5.0, 0.05% Tween 20). The supernatant is then removed. Freshly prepared 100 μL of EDC solution (10 mg / mL, using the above-mentioned MEST solution as a dispersant) and 100 μL of NHS (10 mg / mL, using the above-mentioned MEST solution as a dispersant) are quickly added to the centrifuge tube containing the magnetic beads. Vortex the beads to fully suspend them and activate them at 25°C for 30 min, maintaining the beads in suspension (inversion mixing can be performed using a vertical mixer). After these steps, the carboxyl groups on the surface of the beads are activated and can be covalently coupled to biological ligands with primary amino groups (the activated state should not be stored for long periods of time; immediate coupling is recommended).

[0214] 3. Covalent coupling of different magnetic beads and antibodies

[0215] The antibody dilution solution and the activated carboxyl magnetic beads (see Table 4) were reacted at a molar ratio at 25°C for 2 h, or coupled at 25°C for 1 h and then allowed to stand at 4°C overnight. The magnetic beads were kept suspended during the coupling period (a vertical mixer can be used for inversion mixing). After magnetic separation, the supernatant was aspirated and the remaining antibody content in the supernatant was detected. The amount and concentration of the magnetic bead-coupled antibody were calculated. The magnetic beads were washed two to three times and resuspended in physiological saline to obtain a magnetic bead-antibody conjugate.

[0216] Table 4 Antibody coupling magnetic beads conditions and yields

[0217]

[0218] 4. Incubate at 37°C for 2 hours, separate the magnetic beads using a magnetic stand, and remove the unbound microorganisms and supernatant. Then resuspend the magnetic beads bound to C. difficile with normal saline, use an antibody label removal reagent, 0.05% papain (S10011, Yuanye Bio), and mix with the labeled magnetic beads. Incubate at 37°C for 3 hours to cleave the Fc and Fab of the mouse monoclonal antibody, separating the magnetic beads from C. difficile. Then collect the magnetic beads using a magnetic stand, and the supernatant is the C. difficile suspension. After diluting C. difficile, add it dropwise to a blood cell counting plate and count it under a microscope.

[0219] It can be seen from Table 4 that when the diameter of the carboxyl magnetic beads is 10 μm and the molar ratio of the magnetic beads to the antibody diluent is 1:5, the enrichment effect is the best.

[0220] Example 6 Preparation of magnetic bead-antibody conjugates and enrichment of Clostridium difficile

[0221] 1. Monoclonal Antibody Dilution

[0222] Monoclonal antibody CD1, monoclonal antibody CD2, monoclonal antibody CD3, and monoclonal antibody CD4 were buffer exchanged into 15 mM MES buffer, pH 6.0, and the antibodies were diluted to 2 mg / mL with MES buffer to obtain antibody diluent CD1, antibody diluent CD2, antibody diluent CD3, and antibody diluent CD4, respectively;

[0223] 2. Activation of carboxyl groups on the surface of magnetic beads

[0224] After mixing the magnetic beads, transfer 100 μL of COOH magnetic beads (70113-5, Suzhou Beaver Biotechnology) of different particle sizes to a 1 mL centrifuge tube. Magnetic separation is performed to remove the supernatant, and the beads are magnetically separated and washed twice with 200 μL of MEST solution (100 mM MES, pH 5.0, 0.05% Tween 20). The supernatant is then removed. Freshly prepared 100 μL of EDC solution (10 mg / mL, using the above-mentioned MEST solution as a dispersant) and 100 μL of NHS (10 mg / mL, using the above-mentioned MEST solution as a dispersant) are quickly added to the centrifuge tube containing the magnetic beads. Vortex the beads to fully suspend them and activate them at 25°C for 30 min, maintaining the beads in suspension (inversion mixing can be performed using a vertical mixer). After these steps, the carboxyl groups on the surface of the beads are activated and can be covalently coupled to biological ligands with primary amino groups (the activated state should not be stored for long periods of time; immediate coupling is recommended).

[0225] 3. Covalent coupling of magnetic beads and antibodies

[0226] Take 200ug of antibody dilution and 100μL of the above-mentioned activated carboxyl magnetic beads (diameter 10μm, magnetic beads to antibody molar ratio of 1:5), react at 25°C for 2h, or couple at 25°C for 1h and then stand at 4°C overnight. Keep the magnetic beads suspended during the coupling (vertical mixer can be used for inversion mixing); magnetic separation, aspirate the supernatant and simultaneously detect the remaining antibody content in the supernatant, calculate the amount and concentration of the magnetic bead-coupled antibody, continue to wash the magnetic beads two to three times, and resuspend with physiological saline to obtain magnetic bead antibody conjugate CD1, magnetic bead antibody conjugate CD2, magnetic bead antibody conjugate CD3 and magnetic bead antibody conjugate CD4, respectively.

[0227] 4. Enrichment of Clostridium difficile

[0228] The above four magnetic bead antibody conjugates (magnetic bead antibody conjugate CD1, magnetic bead antibody conjugate CD2, magnetic bead antibody conjugate CD3 and magnetic bead antibody conjugate CD4) were taken and mixed according to the mass ratio of magnetic bead antibody conjugates of 1:1, 1:1:1 or 1:1:1:1 to obtain 11 combinations of CD1+CD2, CD1+CD3, CD1+CD4, CD2+CD3, CD2+CD4, CD3+CD4, CD1+CD2+CD3, CD1+CD2+CD4, CD1+CD3+CD4, CD2+CD3+CD4, and CD1+CD2+CD3+CD4. Take 5ml of stool suspension containing Clostridium difficile (suspend 5g of stool in 5mL of normal saline), add 0.1mg of magnetic bead antibody conjugate CD1, magnetic bead antibody conjugate CD2, magnetic bead antibody conjugate CD3 or magnetic bead antibody conjugate CD4 or the above antibody conjugates, mix and incubate at 37℃ for 2h, separate the magnetic beads using a magnetic stand, and remove the unbound microorganisms and supernatant. Then resuspend the magnetic beads bound to Clostridium difficile with normal saline, use the antibody label removal reagent, that is, 0.05% papain (S10011, Yuanye Bio) and mix with the labeled magnetic beads, incubate at 37℃ for 3h, used to cut the Fc and Fab of the mouse monoclonal antibody, so that the magnetic beads and Clostridium difficile are separated, and then collect the magnetic beads with a magnetic stand. The supernatant is the Clostridium difficile suspension. After diluting Clostridium difficile, add it dropwise to a blood cell counting plate and count it under a microscope.

[0229] Table 5 Antibody-coupled magnetic beads combination types and yields

[0230]

[0231]

[0232] The results are shown in Table 5: The combination of CD1+CD2+CD3+CD4 obtained by mixing the magnetic bead antibody conjugates in a mass ratio of 1:1:1:1 has the highest yield in enriching Clostridium difficile.

[0233] Example 7 Cultivation and Sequencing of Clostridium difficile

[0234] The isolated Clostridium difficile was diluted to 10 3 -10 4The strain was then plated onto CCFA solid medium and incubated anaerobically at 37°C for 48 hours. Colony morphology was observed: the colonies were 3-5 mm in diameter, round, slightly convex, pale yellow, opaque, with irregular edges, and a rough surface. Colonies grown on CCFA plates exhibited yellow-green fluorescence under ultraviolet light. Twenty individual colonies were selected and used as templates for 16S rDNA PCR amplification. Amplified products were separated by agarose gel electrophoresis, revealing a specific band approximately 1500 bp in size, consistent with the expected size. The target band was purified by gel extraction and sequenced. BLAST comparison of the sequencing results against the NCBI database revealed 100% homology between the 16S rDNA gene sequences of the 20 colonies and Clostridium difficile (GenBank: NR_112172.1), confirming that the isolates were Clostridium difficile.

[0235] Although the above embodiments have been described in detail, they are only a part of the embodiments of the present invention, not all of them. People can also obtain other embodiments based on this embodiment without inventiveness, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. A monoclonal antibody against a target protein of Clostridium difficile, characterized in that: The monoclonal antibody is any one of monoclonal antibody CD1, monoclonal antibody CD2, monoclonal antibody CD3 and monoclonal antibody CD4, wherein Monoclonal antibody CD1 includes CD1 heavy chain variable region and CD1 light chain variable region, and their amino acid sequences are: The amino acid sequence of the CD1 heavy chain variable region is shown in SEQ ID NO:9: GGGLVQPGGSLKLSCAASGFDFSRWYKDWVRQAPGKGLEWIGIEPNSD GYNITYSPKLDFIISRDNAKHTLYLQMSKVRSEDTALYYCARLDYTSNSNWG QGTALTVSS, The amino acid sequence of the CD1 light chain variable region is shown in SEQ ID NO: 10: DIVLTQSPATLSVTPGDSVSSLSCWHLSQINSHLGWYQQKSHESPRLLIKV VSKLDSGGIPSRFSGSGSGTDFTLSIDSVETEDFGMYFCWQGWTFGAGTKLE LKRA; Monoclonal antibody CD2 includes CD2 heavy chain variable region and CD2 light chain variable region, and their amino acid sequences are: The amino acid sequence of the CD2 heavy chain variable region is shown in SEQ ID NO: 11: EVKLQESGTVLARPGTSVKMSCRASGYSFYSYWLHWIKQRPGQGLEWV GYGISPASTAPGNSATDKKATLTAVTSASTAYMELSSLTNEDSAVYYCIRGNN YWGQGTTVTVSS, The amino acid sequence of the CD2 light chain variable region is shown in SEQ ID NO: 12: DVVLTQSPLTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLFQRPGQSPK RLIYVVSKLDSGVPDRFYGSGSGTDFTLKISRVEAEDLGVYYCWQGWTFGG GTKLEI; The monoclonal antibody CD3 includes a CD3 heavy chain variable region and a CD3 light chain variable region, and their amino acid sequences are: The amino acid sequence of the CD3 heavy chain variable region is shown in SEQ ID NO: 13: VKPGGSLKLSCAASGFTFSRNDGIWVRQTPDKRLEWVAHIRNDAEGIW MGQKDETRFTISRDQDKQTLYLQMSSLKSEDTAMYYCASTRMADCQMFW GQGTTVTVS, The amino acid sequence of the CD3 light chain variable region is shown in SEQ ID NO: 14: DLVMTQTAPSVPVTPGESVSISCADNCQGEQIKETSTWYWFLQRPGQSP QLLIYHIMGDETGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCARTWGQDT EFGSGTKLEIK; Monoclonal antibody CD4 includes CD4 heavy chain variable region and CD4 light chain variable region, and their amino acid sequences are: The amino acid sequence of the CD4 heavy chain variable region is shown in SEQ ID NO: 15: EVKLVESGPDLVKPSQSLSLTCTVTGYSITTHQPFTWIRQFPGNKLEWMGRADNCQTSMKFTPSWYRISMTRDTSKNQFFLQLNSVTTEDTATYYCARGHTMFTPSEWGQGTSVTVSS The amino acid sequence of the CD4 light chain variable region is shown in SEQ ID NO: 16: DIVLTQSPASSLSASVGEIVTITCRSALYYANENIWYQQKQGKSPQLLVYN LRHGSTGVPSRFSGSGSGTQFSLKINSLQPEDFGSYYCTQDPKDWSLFGGDT KLEIKR; The target protein of monoclonal antibody CD1 is protein TcdA, whose amino acid sequence is shown in SEQ ID NO: 1: KNLHFVWIGGEVSDIALEYIKQWADINAEYNIKLWYDSEAFLVNTLKKAIVESSTTEALQLLEEEIQNPQFDNMKFYKKRMEFIYDRQKRFINYYKSQINKPTVPTIDDIIKSHLVSEYNRDETVLESYRTNSLRKINSNHGIDIRANSLFTEQELLNIYSQELLNRGNLAAASDIVRLLALKNFGG VYLDVDMLPGIHSDLFKTISRPSSIGLDRWEMIKLEAIMKYKKYINNYTSENFDKLDQQLKDNFKLIIESKSEKSEIFSKLENLNVSDLEIKIAFALGSVINQALISKQGSYLTNLVIEQVKNRYQFLNQHLNPAIESDNNFTDTTKIFHDSLFNSATAENSMFLTKIAPYLQVGFMPEARSTISL, The target protein of monoclonal antibody CD2 is protein TcdB, whose amino acid sequence is shown in SEQ ID NO:3: KNLHFIWIGGQINDTAINYINQWKDVNSDYNVNVFYDSNAFLINTLKKTIIESASNDTLESFRENLNDPEFNHTAFFRKRMQIIYDKQQNFINYYKAQKEENPDLIIDDIVKTYLSNEYSKDIDELNAYIEESLNKVTENSGNDVRNFEEFKTGEVFNLYEQELVERWNLAGASDILRVAILKNIGG VYLDVDMLPGIHPDLFKDINKPDSVKTAVDWEEMQLEAIMKYKEYIPEYTSKHFDTLDEEVQSSFESVLASKSDKSEIFLPLGGIEVSPLEVKVAFAKGSIIDQALISAKDSYCSDLLIKQIQNRYKILNDTLGPIISQGNDFNTTMNNFGESLGAIANEENISFIAKIGSYLRVGFYPEANTTITL, The target protein of the monoclonal antibody CD3 is the protein LPxTG, whose amino acid sequence is shown in SEQ ID NO:5: DSTTIQQNKDTLSQIVVFPTGNYDKNEANAMVNRLANIDGKYLNALKQNNLKIKLLSGKLTDEKEYAYLKGVVPKGWEGTGKTWDDVPGLGGSTVALRIGFSNKGKGHDAINLELHETAHAIDHIVLNDISKSAQFKQIFAKEGRSLGNVNYLGVYPEEFFAESFAYYYLNQDTNSKLKSACPQTYSFLQNLAK, The target protein of monoclonal antibody CD4 is protein GDH, whose amino acid sequence is shown in SEQ ID NO:7: MSGKDVNVFEMAQSQVKNACDKLGMEPAVYELLKEPMRVIEVSIPVKMDDGSIKTFKGFRSQHNDAVGPTKGGIRFHQNVSRDEVKALSIWMTFKCSVTGIPYGG GKGGIIVDPSTLSQGELERLSRGYIDGIYKLIGEKVDVPAPDVNTNGQIMSWMVDEYNKLTGQSSIGVITGKPVEFGGSLGRTAATGFGVAVTAREAAAKLGIDMK KAKIAVQGIGNVGSYTVLNCEKLGGTVVAMAEWCKSEGSYAIYNENGLDGQAMLDYMKEHGNLLNFPGAKRISLEEFWASDVDIVIPAALENSITKEVAESIKAK LVCEAANGPTTPEADEVFAERGIVLTPDILTNAGGVTVSYFEWVQNLYGYYWSEEEVEQKEEIAMVKAFESIWKIKEEYNVTMREAAYMHSIKKVAEAMKLRGWY.

2. Use of the monoclonal antibody according to claim 1 in the preparation of magnetic bead-antibody conjugates.

3. A method for preparing a magnetic bead-antibody conjugate, characterized in that: The following steps are involved: 1) diluting the monoclonal antibody according to claim 1 using MES buffer, 2) Activate the carboxyl group of the magnetic beads to obtain activated carboxyl magnetic beads 3) coupling the monoclonal antibody of step 1) with activated carboxyl magnetic beads to obtain a magnetic bead-antibody conjugate, wherein the magnetic bead-antibody conjugate is any one of magnetic bead-antibody conjugate CD1, magnetic bead-antibody conjugate CD2, magnetic bead-antibody conjugate CD3 and magnetic bead-antibody conjugate CD4; The particle size of the activated carboxyl magnetic beads is 10 to 30 μm, and the molar ratio of the monoclonal antibody to the activated carboxyl magnetic beads is 1:5 to 10.

4. The method for preparing the magnetic bead-antibody conjugate according to claim 3, characterized in that: The particle size of the activated carboxyl magnetic beads is 10 μm, and the molar ratio of the monoclonal antibody to the activated carboxyl magnetic beads is 1:

5.

5. Use of an antibody conjugate in enriching or isolating Clostridium difficile, characterized in that: The antibody conjugate is any one or more of the magnetic bead antibody conjugate CD1 prepared by the method of claim 3, the magnetic bead antibody conjugate CD2 prepared by the method of claim 3, the magnetic bead antibody conjugate CD3 prepared by the method of claim 3, and the magnetic bead antibody conjugate CD4 prepared by the method of claim 3.

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