Monoclonal antibody for detecting fetal campylobacter surface protein sapA and application thereof

Abstract

This invention discloses a monoclonal antibody for detecting the surface protein SapA of Campylobacter fetus and its application. The monoclonal antibodies are named 4F6 and 7B3, and the amino acid sequences of the heavy and light chain variable regions of these two antibodies are disclosed. The monoclonal antibody prepared by this invention can specifically recognize the surface protein SapA of Campylobacter fetus. The prepared monoclonal antibody has good specificity, showing no cross-reactivity with non-Camplybacteria such as standard Escherichia coli, Salmonella, Listeria, Klebsiella pneumoniae, Staphylococcus aureus, and Streptococcus, and also with Campylobacter jejuni and Campylobacter coli. The test strip prepared by combining the monoclonal antibody of this invention with colloidal gold can achieve rapid on-site detection of Campylobacter fetus with a low detection limit and high sensitivity, successfully developing an immunological method for the specific detection of Campylobacter fetus.

Description

Technical Field

[0001] This invention relates to the field of immunoassay, specifically to a monoclonal antibody for detecting SapA, a surface protein of Campylobacter fetus, and its application. Background Technology

[0002] Campylobacter fetus subsp. venerealis (Cfv) is the pathogen of bovine reproductive campylobacterial disease (BGC). Wild-type strains of Campylobacter fetus are covered with a high-molecular-weight surface protein, which plays a crucial role in the virulence of the bacterium. This protein has a molecular weight of approximately 97–149 Ku and is essential in causing diseases such as abortion in cattle and sheep. Previous studies have found that the surface protein of *Curculigo fetus* possesses multiple antigenic epitopes and strong pathogenicity, particularly the conserved N-terminus, which may be related to its resistance to the binding of complement component C3b to bacterial cells, excretion, self-assembly, and crystal structure formation. Therefore, it exhibits antiserum bactericidal and anti-phagocytic effects. The high-molecular-weight surface protein is encoded by the Sap gene family and is mainly secreted and transported by the type I secretion system. A polypeptide of 184 amino acids located in the conserved N-terminal region of the surface protein, which binds to IgG antibodies, was screened from two rabbits immunized with *Curculigo fetus* surface protein (97 Ku). Two antigenic epitopes were identified between amino acids 81-110 and 141-160. The complete gene SapA of the *Curculigo fetus* J05577 surface protein consists of 3974 bases, with an open reading frame of 2820 bases. The expressed protein is approximately 97 Ku, consistent with the location of proteins with good immunoprotective effects in the conserved region mentioned above, further indicating that the surface protein is a good diagnostic target.

[0003] Currently, clinical detection of Campylobacter fetus faces a series of challenges. Traditional microbial culture methods, considered the "gold standard" for diagnosis, are extremely cumbersome and time-consuming, rely on complex microaerophilic environments, and are prone to false negatives. While PCR-based molecular detection techniques are fast and highly sensitive, they cannot effectively distinguish between active infectious pathogens and dead bacterial DNA, potentially leading to inaccurate assessment of treatment efficacy and infection status in the later stages of infection or after treatment. Serological methods suffer from insufficient specificity and standardization. In immunological detection, monoclonal antibodies are considered ideal core materials for constructing high-performance diagnostic kits due to their high homogeneity, strong specificity, and the ability to be stably produced in unlimited quantities. However, specific monoclonal antibodies against Campylobacter fetus, especially those capable of recognizing its unique surface antigens and suitable for establishing highly sensitive and specific immunoassay methods, are rarely reported domestically or internationally, and related commercial reagents are scarce. This shortage of crucial reagents severely restricts the development and application of rapid immunological detection technologies (such as lateral flow chromatography strips and quantitative ELISA kits) in the field of Campylobacter fetus detection.

[0004] Therefore, there is an urgent need in this field for specific monoclonal antibodies against Campylobacter fetus, and based on this, to establish a simple, rapid, accurate detection method suitable for field and grassroots use, in order to fill the gap in existing technology and meet the urgent need for efficient diagnostic tools in practical applications. Summary of the Invention

[0005] In view of this, the present invention obtains hybridoma cells that secrete monoclonal antibodies against Campylobacter fetus surface protein SapA, thereby obtaining monoclonal antibodies against Campylobacter fetus surface protein SapA, and establishes a rapid detection method for Campylobacter fetus based on the monoclonal antibody, etc.

[0006] This invention provides a monoclonal antibody for detecting Campylobacter fetus, wherein the monoclonal antibody is selected from either 4F6 or 7B3; wherein the amino acid sequence of the heavy chain complementarity-determining region of the monoclonal antibody 4F6 is as follows: CDR4F6-H1:WVFINQLW; CDR4F6-H2:NLGWWKY; CDR4F6-H3:CRIYYDYPSDYGL; The amino acid sequence of the light chain complementarity-determining region of monoclonal antibody 4F6 is as follows: CDR4F6-L1: QSLLDSDGKTY; CDR4F6-L2: LVS; CDR4F6-L3: WQGKNLPYT; The amino acid sequence of the heavy chain complementarity-determining region of the monoclonal antibody 7B3 is as follows: CDR7B3-H1: GFTFSSYG; CDR7B3-H2: INSNDGRT; CDR7B3-H3: ARGGYGFAY; The amino acid sequence of the light chain complementarity-determining region of monoclonal antibody 7B3 is as follows: CDR7B3-L1: ENIYSN; CDR7B3-L2: GAT; CDR7B3-L3: CQHFWGTPYTF.

[0007] Furthermore, the heavy chain variable region amino acid sequence of the monoclonal antibody 4F6 is SEQ ID NO.1, and the light chain variable region amino acid sequence is SEQ ID NO.5.

[0008] Furthermore, the amino acid sequence of the heavy chain variable region of the monoclonal antibody 7B3 is SEQ ID NO.8, and the amino acid sequence of the light chain variable region is SEQ ID NO.12.

[0009] Furthermore, the monoclonal antibody is capable of specifically recognizing the SapA protein.

[0010] Furthermore, the monoclonal antibody is a mouse-derived IgG1 subtype monoclonal antibody.

[0011] The present invention also provides the use of the monoclonal antibody in the preparation of Campylobacter fetus detection products.

[0012] The present invention provides a reagent or kit for detecting Campylobacter fetus, characterized in that it contains the monoclonal antibody provided by the present invention.

[0013] This invention provides a colloidal gold immunochromatographic test strip for detecting Campylobacter fetus, comprising a sample pad, an absorbent pad, a gold-labeled pad, and a nitrocellulose membrane. The nitrocellulose membrane has detection lines and control lines drawn on it. The sample pad, absorbent pad, gold-labeled pad, and nitrocellulose membrane are all fixed on a base plate. The gold-labeled pad is coated with a monoclonal antibody provided by this invention.

[0014] Preferably, the coating concentration of the monoclonal antibody is 6 μg / mL.

[0015] Beneficial effects: This invention, for the first time, isolated and screened two hybridoma cell lines capable of stably secreting high-titer, highly specific antibodies against SapA. The monoclonal antibodies secreted by these cells, targeting a specific epitope of this antigen, were named 4F6 and 7B3, respectively, and the amino acid sequences of the heavy and light chain variable regions of these two antibodies were disclosed. Experiments showed that both monoclonal antibodies specifically recognized the SapA protein in Western blot analysis, with both antibody subclasses being IgG1, and the antibody titers of both reaching 1:409600. The monoclonal antibodies of this invention are produced by specific single cell lines and possess high specificity. They show no cross-reactivity with standard non-Camplybacteria such as *Escherichia coli*, *Salmonella*, *Listeria*, *Klebsiella pneumoniae*, *Staphylococcus aureus*, and *Streptococcus*, and also with Campylobacter jejuni and Campylobacter coli. Based on this invention, a rapid detection immunochromatographic test strip for Campylobacter fetus, constructed with a monoclonal antibody that can recognize the SapA surface protein of *Camplylobacter fetus*, is simple to operate, provides clear and distinguishable results, and exhibits high sensitivity and specificity, making it suitable for rapid real-time detection of *Camplylobacter fetus*. Attached Figure Description

[0016] Figure 1 SDS-PAGE electrophoresis image of Campylobacter fetus SapA protein, where M: protein molecular weight standard; 1: flow-through buffer; 2: purified protein; Figure 2 Western blot electrophoresis image of Campylobacter fetus SapA protein; Figure 3 SDS-PAGE electrophoresis images of Campylobacter fetus monoclonal antibodies 4F6 and 7B3; Figure 4 The results of subtype identification of Campylobacter fetus monoclonal antibodies 4F6 and 7B3; Figure 5 The results show the titer of monoclonal antibodies 4F6 and 7B3 against Campylobacter fetus. Figure 6 PCR identification results of the variable regions of Campylobacter fetus monoclonal antibodies 4F6 and 7B3; Figure 7 Colloidal gold color diagram showing the optimal pH value and the best amount of conjugated antibody for Campylobacter fetus immunochromatographic test strips; Figure 7 In A, 1 represents the unadjusted pH value; values ​​2 through 10 represent pH values ​​of 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, and 9.5, respectively. Figure 7 In B, the amounts of conjugated antibodies 1 to 8 were 0 μg / mL, 2 μg / mL, 4 μg / mL, 6 μg / mL, 8 μg / mL, 10 μg / mL, 15 μg / mL, and 20 μg / mL, respectively. Figure 8Determination of antibody concentration embedded in the C-line of the Campylobacter fetus immunochromatographic test strip; Figure 9 Determination of antibody concentration embedded in the T line of Campylobacter fetus immunochromatographic test strips; Figure 10 This is a diagram showing the specific results of the Campylobacter fetus immunochromatographic test strip; Figure 11 The image shows the sensitivity results of PCR detection of Campylobacter fetus. Figure 12 The sensitivity results of the Campylobacter fetus immunochromatographic test strip are shown in the figure. Figure 13 The graph shows the intra-batch repeatability results of the Campylobacter fetus immunochromatographic test strip; Figure 14 Figure showing the batch-to-batch repeatability results of the Campylobacter fetus immunochromatographic test strip. Detailed Implementation

[0017] The embodiments of the present invention will be described in detail below with reference to specific examples. These examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Rather, they should be understood as a more detailed description of certain aspects, characteristics, and implementation schemes of the invention. Unless otherwise specified, the instruments, reagents, and materials used in the following examples are commercially available, and the experimental methods are conventional methods in the art.

[0018] Example 1: Affinity Purification and Identification of Recombinant Protein (1) Induced expression of recombinant protein: The NCBI accession number for the nucleotide sequence of the Campylobacter fetus protein encoding gene is AY211269.1, and the accession number for the corresponding amino acid sequence is AAO64227.1. First, the target gene SapA was cloned into the pET28a(+) vector by enzyme digestion and ligation to construct the recombinant plasmid pET28a(+)-SapA. The recombinant plasmid pET28a(+)-SapA was transformed into Escherichia coli BL21(DE3) competent cells and evenly spread on kanamycin-selective LB solid plates and incubated upside down at 37℃ for 12 h. Single colonies were picked and cultured in kanamycin-selective LB liquid medium at 37℃ with shaking at 180 rpm until the bacterial culture OD 600 When the concentration was 0.8, IPTG was added to a final concentration of 24 mg / mL and induced at 37℃ for 6 h.

[0019] (2) Recombinant protein expression identification and solubility analysis: 800 mL of induced bacterial culture was centrifuged at 10,000 rpm for 3 min at 4℃, the supernatant was discarded, and the bacterial cells were collected and resuspended in 15 mL PBS. The cells were then sonicated on ice using the following sonication program: 5 s sonication, 5 s interval, 35% power. After sonication, the bacterial culture was centrifuged at 8,000 rpm for 5 min at 4℃ to separate insoluble components. 20 μL of the supernatant and precipitate were added to 5× protein loading buffer and heated at 100℃ for 10 min. The bacterial culture samples before and after induction, as well as the supernatant and precipitate samples after sonication, were identified by SDS-PAGE. Results are shown below. Figure 1 This indicates successful purification of the recombinant SapA protein. The purified SapA protein was then identified by Western blot using a His-tagged antibody. The results are shown in [Figure number missing]. Figure 2 .

[0020] Example 2: Indirect ELISA Antibody Detection Method Based on SapA Protein (1) Antigen coating: Coat the SapA antigen at 2.5 μg / mL onto the microplate, 100 μL / well, and incubate overnight at 4°C; discard the liquid in the well, wash 3 times with PBST, discard the washing solution and pat dry; (2) Blocking: Block with 5% BSA, 200 μL / well, incubate at 37℃ for 30 min, discard the blocking solution, wash 3 times with PBST, discard the washing solution and pat dry; (3) Incubation of primary antibody: Dilute positive serum (serum of mice injected with immunogen) and negative serum (serum of normal mice not injected with immunogen) with PBST at a ratio of 1:500, 100 μL / well, incubate at 37℃ for 30 min, discard the primary antibody, wash 3 times with PBST, discard the washing solution and pat dry. (4) Incubation of secondary antibody: Add goat anti-mouse secondary antibody (1:4000 dilution), 100 μL / well, incubate at 37℃ for 1 h 30 min, discard the primary antibody, wash 5 times with PBST, discard the washing solution and pat dry; (5) Color development: Add TMB color development solution, 100 μL / well, and incubate at 37℃ for 20 min; (6) Termination: Add stop solution, 50 μL / well, and measure OD using a microplate reader. 450nm The value.

[0021] Example 3: Preparation of monoclonal antibody based on Campylobacter fetus surface protein SapA and screening of hybridoma cells (1) Immunizing mice The recombinant protein prepared in Example 1 (the prepared SapA protein) and Freund's complete adjuvant were mixed in a 1:1 ratio and placed on ice. The mixture was then emulsified for approximately 5 minutes using an emulsifier. A drop of the emulsifier should not spread when dropped into water, indicating sufficient emulsification. This mixture was then used as an immunogen to immunize 6-week-old female BALB / c mice (0.2 ml / mouse). Fourteen days later, the antigen was emulsified with Freund's incomplete adjuvant, and two booster immunizations were performed, each two weeks apart. One week after the completion of the three immunizations, blood was collected from the intertail vein of the mice. After centrifugation, the supernatant was used to detect the serum antibody titer of the mice via indirect ELISA. Three days before fusion, mice were intraperitoneally injected with the immunogen for a pulse immunization.

[0022] (2) Preparation of myeloma cells SP2 / 0 myeloma cells: SP2 / 0 cells stored in liquid nitrogen tanks are revived and passaged. After passage to 5-10 flasks, the cells are growing well, are round, clear, uniform in size, neatly arranged, and undergoing logarithmic division, ready for fusion.

[0023] (3) Preparation of feeder cells One 4-5 week old female Balb / C mouse was euthanized by cervical dislocation and then immersed in a beaker containing 75% alcohol for 5 minutes for sterilization. The mouse was fixed in a laminar flow hood, and the abdominal skin was opened with sterile scissors to expose the peritoneum. The peritoneum was lifted with forceps, and 10 mL of RPMI-1640 complete culture medium was slowly injected into the mouse abdomen using a sterile syringe. The culture medium containing macrophages was repeatedly aspirated from both sides of the abdominal cavity, centrifuged, and the supernatant was discarded. The spleen of the mouse was then removed, placed in a cell strainer, crushed, and rinsed with RPMI-1640 complete culture medium. The rinsed spleen cells were collected in a centrifuge tube, centrifuged, and the supernatant was discarded. The macrophages and spleen cells were mixed and added to 50 mL of HAT medium. The mixture was thoroughly mixed, plated into 5 96-well cell culture plates, and placed in a CO2 incubator at 37°C overnight for later use.

[0024] (4) Preparation of spleen cells After euthanizing the mouse with the highest titer in step (1) by enucleation, the mouse was immersed in a beaker containing 75% alcohol for 5 minutes for sterilization. The mouse was then fixed in a laminar flow hood, and the abdominal skin was opened with sterile scissors and a spatula to remove the spleen. Excess tissue was removed with forceps, and the spleen was then ground up to collect spleen cells.

[0025] (5) Cell fusion Prepared SP2 / 0 cells and spleen cells from immunized Balb / C mice were fused at a ratio of 1:10 using 1 mL of PEG1500 in a 37°C water bath. The cells were then plated and cultured in 96-well plates at 37°C. After 5 days, half the medium was replaced with HAT medium, and after 10 days, the entire medium was replaced with HT medium.

[0026] (6) Detection and screening of positive wells The enzyme-labeled plate coated with Campylobacter fetus SapA antigen was removed, and the culture supernatant of the fusion cells was detected using the indirect ELISA detection method established in Example 2. Strongly positive wells were used for subcloning, and a total of 3 subclonings were performed. After the last subcloning, the antibody titer of the hybridoma cell supernatant was detected, and those that could stably secrete antibodies were screened for large-scale amplification for subsequent experiments.

[0027] Example 4: Identification based on monoclonal antibody against Campylobacter fetus surface protein SapA (1) Preparation of monoclonal antibody ascites: The preparation of ascites using monoclonal antibody-induced ascites was carried out via an in vivo induction method, as follows: Female Balb / C mice aged 10-12 weeks were intraperitoneally injected with liquid paraffin, 0.5 mL per mouse. One week after injection, the hybridoma cells in the logarithmic growth phase obtained in Example 3 were gently washed twice with sterile PBS, resuspended in 1640 complete culture medium, and counted. The cell concentration was adjusted to 1×10⁻⁶. 7 The dose was administered via intraperitoneal injection, with each mouse receiving 0.5 mL. After one week, when mice showed obvious abdominal enlargement, disheveled fur, and difficulty moving, ascites fluid could be collected, the supernatant was centrifuged and collected, and stored at -80℃.

[0028] (2) Purification of monoclonal antibodies: Filter the ascites fluid using a 0.45 μm sterile filter and collect the filtrate. Add 1 mL of Protein A+G agarose gel to a chromatography column (FCL03 Beyotime affinity chromatography column, 1 mL empty column tube). After the agarose gel precipitates, equilibrate the column with 5–10 column volumes of binding buffer (PBS). Add the filtrate sequentially to the equilibrated column in 500 μL increments, allowing it to elute slowly, and collect the eluent. Elute non-specifically adsorbed proteins with 10 column volumes of binding buffer and collect the eluent. Elute the protein with 10 column volumes of elution buffer (20 mM glycine, pH 2.7), and add 100 μL of neutralization buffer (1 mol / L Tris HCl) per mL of eluent to the collection tube to neutralize the pH of the eluent. This eluent is the purified monoclonal antibody. After sample purification, the resin was washed sequentially with 3 column volumes of binding buffer, 5 column volumes of deionized water, and 5 column volumes of 25% ethanol. Then, the same column volume of 25% ethanol was added, and the column was stored at 4°C. Western blot analysis was performed on the purified monoclonal antibodies 4F6 and 7B3 with SapA protein to verify their reactivity. The results showed that both monoclonal antibodies 4F6 and 7B3 were reactivity-positive, with 4F6 exhibiting stronger reactivity. Figure 3 ).

[0029] (3) Subtype identification The monoclonal antibodies were identified according to the instructions of the mouse monoclonal antibody subtype identification kit, using the following method: Remove the kit from the 4°C freezer and equilibrate at room temperature for 30 min. Use a pipette to directly aspirate the cell culture supernatant from the cell culture plate, adding 50 μL each of the cell supernatant and sample diluent to the wells of the strip. Incubate for 30 min. Wash the plate 5 times and tap to dry any remaining liquid. Add 50 μL of each different subtype standard to each well. Gently tap the sides of the plate holder to mix. Cover the plate with sealing film and incubate at 37°C for 30 min. Wash the plate 5 times and tap to dry any remaining liquid. Add 50 μL each of colorimetric solutions A and B to each well. Develop at room temperature in the dark for 10–20 min, then add 50 μL of stop solution to each well to stop the reaction. The colorimetric results are shown below. Figure 4 As shown, based on visual inspection, the 4F6 and 7B3 antibody subclasses were determined to be IgG1.

[0030] (4) Valence assessment The activity of two monoclonal antibodies was identified using the indirect ELISA antibody detection method established in Example 2. The titers of both 4F6 and 7B3 antibodies reached 1:409600. Figure 5 ).

[0031] Example 4: Sequencing of the heavy and light chain variable regions of two Campylobacter fetus SapA monoclonal antibodies. (1) RNA extraction from hybridoma cells (TRIzol method): Total RNA was extracted from hybridoma cells using the Kangwei Century Biotechnology Co., Ltd. Ultrapure RNA Extraction Kit (Catalog No.: CW0581S) and stored at -70℃.

[0032] (2) Reverse transcription: Reverse transcription was performed using the one-step reverse transcription kit from Tiangen Biotech (Beijing) Co., Ltd.

[0033] (3) Amplification of the variable regions of heavy and light chains: The variable regions of heavy and light chains of monoclonal antibodies were amplified using the primers shown in Table 1; the electrophoresis products of this region were excised and recovered, purified using a kit, and then sent for sequencing.

[0034] Table 1. PCR primers for amplifying the variable regions of the heavy and light chains of monoclonal antibodies.

[0035] (4) The PCR amplification results of the variable regions of the heavy and light chains of the two monoclonal antibodies are shown in the figure. Figure 6 .

[0036] Annotation of the complementarity-determining region (CDR) of the heavy and light chains: The amino acid sequences of the variable regions obtained from sequencing were imported into a prediction website (https: / / www.imgt.org / IMGT_vquest / input) to analyze the constituent elements of the variable regions of the heavy and light chains of the two monoclonal antibodies. The results are as follows.

[0037] The amino acid sequence of the variable region of the 4F6 heavy chain of the monoclonal antibody is as follows: QVQSAGVWTWPGGALTEPVHHLHCLWVFINQLWCTLGSPASRKGSGVAGSNLGWWKYKLFGSHVQTEHXQRQLQEPSFLKNEQSABHSHVLLCRIYYDYPSDYGLLGSRNLSHRLR (SEQ ID NO.1) The amino acid sequences of the complementarity-determining region of the 4F6 heavy chain of the monoclonal antibody are as follows: CDR4F6-H1:WVFINQLW (SEQ ID NO.2) CDR4F6-H2:NLGWWKY (SEQ ID NO.3) CDR4F6-H3:CRIYYDYPSDYGL (SEQ ID NO.4) The amino acid sequence of the variable region of the light chain of monoclonal antibody 4F6 is as follows: TQSPLTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLVSNLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGKNLPYTFGGGTKLEIK (SEQ ID NO.5) The amino acid sequences of the complementarity-determining region of the light chain of monoclonal antibody 4F6 are as follows: CDR4F6-L1:QSLLDSDGKTY (SEQ ID NO.6) CDR4F6-L2:LVS CDR4F6-L3:WQGKNLPYT (SEQ ID NO.7) The amino acid sequence of the variable region of the heavy chain of monoclonal antibody 7B3 is as follows: VESGGGLVQPGGSLKLSCAASGFTFSSYGMSWVRQTPDKRLELVATINSNDGRTFYPDSVKGRFTISRDDNAKNTLYLQMSGLKSEDTAMFYCARGGYGFAYWGQGTLVTV (SEQ ID NO.8) The amino acid sequences of the complementarity-determining region of the heavy chain of monoclonal antibody 7B3 are as follows: CDR7B3-H1:GTFFSSYG (SEQ ID NO.9) CDR7B3-H2:INSNDGRT (SEQ ID NO.10) CDR7B3-H3:ARGGYGFAY(SEQ ID NO.11) The amino acid sequence of the variable region of the light chain of monoclonal antibody 7B3 is as follows: IQLTQSPASLSVSVGETVTITCRASENIYSNLAWYQQKQGKSPQLLVYGATNLADGVPSRFSGSGSGTQYSLKINSLQSEDFGSYYCQHFWGTPYTFGGGTK (SEQ ID NO.12) The amino acid sequences of the complementarity-determining region of the light chain of monoclonal antibody 7B3 are as follows: CDR7B3-L1:ENIYSN (SEQ ID NO.13) CDR7B3-L2:GAT CDR7B3-L3:CQHFWGTPYTF (SEQ ID NO.14) Example 5: Preparation method of Campylobacter fetus immunochromatographic test strip The application of the monoclonal antibody of this invention is illustrated by taking the establishment of a colloidal gold detection method using monoclonal antibody 4F6 as an example.

[0038] (1) The preparation method of gold-labeled antibody conjugate and the treatment method of gold-labeled pads are as follows: Take 30 mL of colloidal gold with a particle size of 50 nm, add 2% K2CO3 to adjust the pH to 7.5, then add anti-Campylobacter fetus monoclonal antibody 4F6, react at 37℃ for 1 h, add 3 mL of 10% BSA, block for 1 h, then centrifuge at 3000 rpm for 20 min at 4℃, collect the precipitate and resuspend the precipitate with a reconstitution solution to obtain the gold-labeled antibody conjugate. Take the same volume of gold-labeled antibody conjugate and uniformly adsorb it onto the same volume of gold-labeled pad, dry it and store it for later use.

[0039] (2) Preparation of nitrocellulose membrane with detection line and quality control line: The prepared polyclonal antibody against Campylobacter fetus specific antigen SapA and goat anti-mouse-IgG were uniformly sprayed onto the nitrocellulose membrane (NC membrane) at a concentration of 1uL / cm using a membrane scrubbing instrument and dried in a constant temperature oven at 37℃ for later use.

[0040] (3) Sample pad treatment: The sample pad is where the test strip is added to add the test sample. The sample pad is treated with some buffer solution to improve the binding and release of the test sample and the gold-labeled antibody on the gold-labeled pad. It is pretreated with a mixture of 0.2% Tween-20 and 3% sucrose.

[0041] (4) Assembly of the test strip: Attach the sample pad, gold label pad, nitrocellulose membrane, and absorbent pad to the plastic base plate from top to bottom to assemble the test strip. The absorbent pad is attached to the right end of the nitrocellulose membrane, overlapping by 0.2 mm. The gold label pad is attached to the left end of the nitrocellulose membrane, overlapping by 0.2 mm. The left end of the gold label pad is attached to the sample pad, overlapping by approximately 2 mm. Finally, cut the strip and store it for later use.

[0042] Example 6: Parameter Optimization and Determination of Campylobacter fetus Immunochromatographic Test Strip (1) Exploration of the optimal pH value for colloidal gold-labeled monoclonal antibodies Ten sample wells of a removable 96-well plate were used. 0.3 mL of 50 nm colloidal gold solution was added to each well. The pH of the colloidal gold solution in each well was adjusted using 2% K₂CO₃ to achieve pH values ​​of 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, and 9.5, respectively. One tube served as a control group, without treatment. Campylobacter fetus monoclonal antibody 4F6 was adjusted to 1 mg / mL, and 10 μL was added to each well. After mixing, the mixture was incubated at 37°C for 1 h. Then, 30 μL of 10% NaCl was added, and the mixture was incubated at 37°C for 10 min, followed by 2 h at room temperature. The color change of the colloidal gold solution was observed, and the optimal pH value was determined to be 7.5 (see [link to relevant documentation]). Figure 7 A).

[0043] (2) Exploration of the optimal concentration of colloidal gold-labeled monoclonal antibody Eight sample wells of a removable 96-well plate were used. 0.3 mL of 30 nm colloidal gold solution was added to each well. After adjusting the pH of the colloidal gold solution to the optimal value, different volumes of monoclonal antibody were added to centrifuge tubes to obtain concentrations of 0 μg / mL, 2 μg / mL, 4 μg / mL, 6 μg / mL, 8 μg / mL, 10 μg / mL, 15 μg / mL, and 20 μg / mL. The mixture was thoroughly mixed and incubated at 37°C for 1 hour. Then, 30 μL of 10% NaCl solution was added, and the mixture was allowed to stand at room temperature for 1 hour. The optimal binding of the monoclonal antibody to the colloidal gold particles was determined to be when the colloidal gold sol showed no color change. Therefore, the optimal concentration of the colloidal gold-labeled monoclonal antibody was determined to be 6 μg / mL (see [link to article]). Figure 7 B).

[0044] (3) Determination of antibody concentration for C-line embedding Based on the above optimization results, commercially available goat anti-mouse IgG was diluted to 2.0 mg / mL, 1.5 mg / mL, 1.0 mg / mL, 0.5 mg / mL, and 0.1 mg / mL, respectively, and sprayed onto line C. After drying, PBS was added to the sample pad. During the parameter optimization phase, the results were judged based on three criteria: whether the test strip bands showed color, the intensity of the color, and the cleanliness of the background. The optimal concentration of goat anti-mouse IgG antibody for embedding on line C was ultimately determined to be 1.0 mg / mL. Figure 8 ).

[0045] (4) Selection of optimal antibody coating concentration for T-line detection The treated NC membrane was selected, and the prepared polyclonal antibody was used as the detection line. The concentration of Campylobacter fetus polyclonal antibody was adjusted to 2.0 mg / mL, 1.5 mg / mL, 1.25 mg / mL, 1.0 mg / mL, 0.75 mg / mL, 0.5 mg / mL, 0.25 mg / mL, 0.1 mg / mL, and 0 mg / mL for streaking. Positive test samples were added, and based on the test results, the optimal antibody concentration for the detection line was determined to be 1.5 mg / mL. Figure 9 ).

[0046] Example 7: Performance Testing of the Campylobacter fetus Immunochromatographic Test Strip 1. Specific detection Test strips were prepared according to the optimized parameters of Example 6. Bacterial suspensions (10 μL each) of Campylobacter (Campylobacter coli, Campylobacter jejuni, Campylobacter fetus) and non-Campylobacter (Standard Escherichia coli, Salmonella, Listeria, Klebsiella pneumoniae, Staphylococcus aureus, Streptococcus) were respectively prepared. 9(CFU / mL) was added to the sample pad of the test strip for detection. The color development of the T / C lines was observed. If both the T and C lines were visible, the result was positive; if only the C line was visible, the result was negative. The results showed that only the Campylobacter fetus suspension produced a positive result. Figure 10 ).

[0047] 2. Sensitivity Detection To determine the limit of detection (LOD) for Campylobacter fetus on the test strip, the pure culture of Campylobacter fetus standard strain (BNCC360236) was diluted with sterile PBS buffer to the following 10 bacterial concentrations: 1×10⁻⁶. 9 CFU / mL, 1×10 8 CFU / mL, 1×10 7 CFU / mL, 1×10 6 CFU / mL, 1×10 5 CFU / mL, 1×10 4 CFU / mL, 1×10 3 CFU / mL, 1×10 2 CFU / mL, 1×10 1 CFU / mL, 1×10 0 CFU / mL, bacterial suspensions of each concentration were added for testing. After shaking and mixing the bacterial suspension, 100 μL was directly added to the sample pad of the test strip (according to the optimized test strip detection system in Example 6). Simultaneously, the same volume of bacterial suspension from each gradient was used as a template for PCR detection. The test results showed that the detection concentration of Campylobacter fetus bacterial suspension was 1 × 10⁻⁶ CFU / mL. 9 CFU / mL ~ 1×10 2 CFU / mL Figure 11 When the bacterial concentration of the pure culture suspension of Campylobacter fetus is 1×10⁻⁶ 9 CFU / mL ~ 1×10 2 At CFU / mL, the color of the T-band weakens as the bacterial suspension concentration decreases; therefore, the minimum detectable concentration of Campylobacter fetus bacterial suspension on the test strip is 1×10⁻⁶. 2 CFU / / mL ( Figure 12 It has a sensitivity comparable to PCR detection.

[0048] 3. Repeatability testing The Campylobacter fetus immunochromatographic test strip prepared in this invention was subjected to intra-batch repeatability testing. The same batch of test strips was used to test the same Campylobacter fetus bacterial suspension sample at different time points. Figure 13 Samples of bacteria 1, 3, 5, and 7 in the sample and unrelated bacterial cultures ( Figure 13(2, 4, 6, 8); inter-batch repeatability tests were also performed, using test strips prepared from different batches to test the same Campylobacter fetus bacterial suspension samples ( Figure 14 Samples of bacteria 1, 3, 5, and 7 in the sample and unrelated bacterial cultures ( Figure 14 (2, 4, 6, 8). The results showed that the prepared colloidal gold test strips had good intra-batch repeatability ( Figure 13 ) and batch repeatability ( Figure 14 All are relatively good.

[0049] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A monoclonal antibody for detecting Campylobacter fetus, characterized in that, The monoclonal antibody is selected from either 4F6 or 7B3; wherein, the amino acid sequence of the heavy chain complementarity-determining region of the monoclonal antibody 4F6 is as follows: CDR4F6-H1:WVFINQLW; CDR4F6-H2:NLGWWKY; CDR4F6-H3:CRIYYDYPSDYGL; The amino acid sequence of the light chain complementarity-determining region of monoclonal antibody 4F6 is as follows: CDR4F6-L1: QSLLDSDGKTY; CDR4F6-L2: LVS; CDR4F6-L3: WQGKNLPYT; The amino acid sequence of the heavy chain complementarity-determining region of the monoclonal antibody 7B3 is as follows: CDR7B3-H1: GFTFSSYG; CDR7B3-H2: INSNDGRT; CDR7B3-H3: ARGGYGFAY; The amino acid sequence of the light chain complementarity-determining region of monoclonal antibody 7B3 is as follows: CDR7B3-L1: ENIYSN; CDR7B3-L2: GAT; CDR7B3-L3: CQHFWGTPYTF.

2. The monoclonal antibody as described in claim 1, characterized in that, The amino acid sequence of the heavy chain variable region of the monoclonal antibody 4F6 is SEQ ID NO.1, and the amino acid sequence of the light chain variable region is SEQ ID NO.

5.

3. The monoclonal antibody as described in claim 1, characterized in that, The amino acid sequence of the heavy chain variable region of the monoclonal antibody 7B3 is SEQ ID NO.8, and the amino acid sequence of the light chain variable region is SEQ ID NO.

12.

4. The monoclonal antibody as described in claim 1, characterized in that, The monoclonal antibody can specifically recognize the SapA protein.

5. The monoclonal antibody as described in claim 1, characterized in that, The monoclonal antibody is a mouse-derived IgG1 subtype monoclonal antibody.

6. The use of the monoclonal antibody according to any one of claims 1 to 5 in the preparation of Campylobacter fetus detection products.

7. A reagent or kit for detecting Campylobacter fetus, characterized in that, It contains the monoclonal antibody as described in any one of claims 1 to 5.

8. A colloidal gold immunochromatographic test strip for detecting Campylobacter fetus, comprising a sample pad, an absorbent pad, a gold-labeled pad, and a nitrocellulose membrane, wherein the nitrocellulose membrane has detection lines and control lines drawn on it, and the sample pad, absorbent pad, gold-labeled pad, and nitrocellulose membrane are all fixed to a base plate, characterized in that, The gold-labeled pad is coated with the monoclonal antibody as described in any one of claims 1 to 5.

9. The colloidal gold immunochromatographic test strip as described in claim 8, characterized in that, The coating concentration of the monoclonal antibody is 6 μg / mL.

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