Monoclonal antibody-based salmonella colloidal gold immunochromatography test strip and preparation method thereof

Abstract

The invention relates to the technical field of biological detection, and discloses a salmonella monoclonal antibody, a salmonella colloidal gold immunochromatography test strip based on the antibody and a preparation method of the salmonella colloidal gold immunochromatography test strip. Salmonella is used as an antigen, a polyclonal antibody and a monoclonal antibody are prepared respectively, the monoclonal antibody 8E71C8 is screened out, and the salmonella colloidal gold immunochromatography test strip is prepared. The purified monoclonal antibody is used as a 30 nm colloidal gold marker, the optimal pH and the optimal antibody labeling amount of binding of the monoclonal antibody and colloidal gold and the concentration of the polyclonal antibody used as a detection line are optimized, the prepared salmonella colloidal gold immunochromatography test strip is high in specificity, the sensitivity is about 1 * 10 < 4 > CFU / mL and is higher than the average detection lower limit of domestic similar products, and the salmonella colloidal gold immunochromatography test strip can be used for detecting salmonella. And the test strip is simple to operate and high in efficiency, can be used as a means for rapidly and visually detecting the salmonella, and has popularization and application values.

Description

Technical Field

[0001] This invention relates to the field of biological detection technology, specifically to a Salmonella monoclonal antibody, a Salmonella colloidal gold immunochromatographic test strip based on the monoclonal antibody, and a method for preparing the same. Background Technology

[0002] In recent years, colloidal gold immunochromatographic test strips have been increasingly used for the detection of Salmonella. Monoclonal antibodies have high specificity, stability and reproducibility. Therefore, compared with other detection methods, the Salmonella colloidal gold immunochromatographic test strip method based on monoclonal antibodies has the advantages of low cost, simple operation and suitability for non-professionals.

[0003] However, the detection sensitivity of colloidal gold immunochromatographic test strips for Salmonella is generally not high enough. For example, the literature retrieved, "Liu Jingwu, Fu Sulan, Ren Tianhong, et al. Study on detection of Salmonella in feces of patients with diarrhea by colloidal gold immunochromatographic test strips [J]. Chinese Journal of Health Laboratory Technology, 2014, 24(01):66-67", mentions that the detection limit of this test strip for Salmonella in feces is 10. 5 CFU / mL; For example, in the Chinese invention patent application CN114720690A, published on July 8, 2022, entitled "A Method for Preparing a Colloidal Gold Immunochromatographic Test Strip for Salmonella Enteritis," paragraph

[0044] mentions that the test strip prepared by this invention has a sensitivity of 1×10⁻⁶ CFU / mL; 6 CFU / mL. Summary of the Invention

[0004] To solve the above-mentioned technical problems, the present invention has prepared a Salmonella monoclonal antibody and a Salmonella colloidal gold immunochromatographic test strip based on the monoclonal antibody.

[0005] First, the Salmonella monoclonal antibody provided by this invention is named 8E7 1C8. The monoclonal antibody, named 8E7 1C8, has a heavy chain variable region comprising three complementarity-determining regions: VH-CDR1, VH-CDR2, and VH-CDR3, wherein: The amino acid sequence of VH-CDR1 is GFTFTDYY; The amino acid sequence of VH-CDR2 is IRNKANGYTT; The amino acid sequence of VH-CDR3 is ARPLYGNYWYFDV; The light chain variable region of the monoclonal antibody 8E7 1C8 includes three complementarity-determining regions: VL-CDR1, VL-CDR2, and VL-CDR3, wherein: The amino acid sequence of VL-CDR1 is SVDSYGNSF; The amino acid sequence of VL-CDR2 is RAS; The amino acid sequence of VL-CDR3 is QQNNEDPWT.

[0006] Specifically, the amino acid sequence of the heavy chain variable region of the monoclonal antibody 8E7 1C8 is SEQ ID NO:2.

[0007] Specifically, the amino acid sequence of the light chain variable region of the monoclonal antibody 8E7 1C8 is SEQ ID NO:4.

[0008] Specifically, the heavy chain constant region of the monoclonal antibody 8E7 1C8 is of the IgG2b type.

[0009] This invention also provides the application of monoclonal antibody 8E7 1C8 in the preparation of Salmonella detection products.

[0010] The present invention also provides a reagent or kit for detecting Salmonella containing monoclonal antibody 8E7 1C8.

[0011] The present invention also provides a colloidal gold immunochromatographic test strip for detecting Salmonella, comprising a sample pad, an absorbent pad, a gold-labeled pad, and a nitrocellulose membrane (NC membrane). The nitrocellulose membrane has a detection line (T line) and a control line (C line). 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 monoclonal antibody 8E7 1C8.

[0012] Specifically, the coating concentration of the monoclonal antibody 8E7 1C8 is 7.2 μg / mL.

[0013] Specifically, the detection line is coated with Salmonella polyclonal antibody at a concentration of 2 mg / mL.

[0014] The present invention also provides a method for preparing the above-mentioned Salmonella monoclonal antibody, comprising the following steps: (a) Immunization procedure: BALB / c mice were immunized with Salmonella as an immunogen at a dose of 50 μg / mouse. (b) Screening before fusion: Mice with serum titers ≥1:16000 were screened by indirect ELISA after immunization, and their spleen cells were fused with myeloma cells mediated by a chemical fusion agent. (c) Cell screening and line establishment: Salmonella was used as an antigen to screen positive hybridoma cells. After subcloning, cell lines with heavy chain constant region of IgG2b type and CDR region sequence of secreting antibody conforming to SEQ ID NO:5~10 were screened to obtain monoclonal hybridoma cell lines that can stably secrete the monoclonal antibody 8E7 1C8. (d) Antibody acquisition and quality control: The above cell lines were cultured, and antibodies were produced by ascites fluid preparation or cell culture. After affinity chromatography purification, the specific binding and titer of the antibodies to Salmonella were verified.

[0015] The monoclonal antibody 8E7 1C8 prepared in this invention has the following beneficial effects: 1) High specificity: By accurately identifying Salmonella antigenic epitopes through the CDR region, Salmonella can be effectively distinguished from other foodborne pathogens; 2) The Salmonella colloidal gold immunochromatographic test strip based on this monoclonal antibody has a sensitivity of up to 1×10⁻⁶. 4 CFU / mL, higher than the sensitivity of similar domestic products; 3) The complete sequence of the core variable region and CDR region is disclosed. Combined with the known IgG2b constant region sequence, those skilled in the art can quickly achieve antibody cloning, expression and preparation. 4) Wide range of applications: In addition to being used to develop the colloidal gold immunochromatographic test strips provided by this invention, it can also be used to develop various products such as Salmonella detection kits, immunomagnetic beads, and antibody chips, which are suitable for rapid screening of Salmonella in food, environment, and clinical samples. Attached Figure Description

[0016] Figure 1 This is the result of Salmonella titer detection after four immunizations of mice in the antiserum titer detection process of this invention; Figure 2 The titer test results of the antibodies produced by cell line 8E7 1C8 in this invention; Figure 3 This is an electrophoresis diagram for purity detection of the 8E7 1C8 monoclonal antibody in this invention, where M is a protein marker and 1 is the purified antibody from the 8E71C8 cell line. Figure 4 The results show the identification of the optimal pH value for the colloidal gold-labeled 8E7 1C8 monoclonal antibody in this invention, where 1 represents the unadjusted pH value; and 2 to 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 5 The results show the identification of the optimal coating amount of the colloidal gold-labeled 8E7 1C8 monoclonal antibody in this invention, wherein the coating amounts of 1 to 8 are 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 6 The results show the determination of the optimal coating concentration of polyclonal antibody for T-line detection of Salmonella in this invention, wherein the T-line concentrations for 1-4 are 2.0 mg / mL, 1.5 mg / mL, 1.0 mg / mL, and 0.5 mg / mL, respectively. Figure 7 The results of the specific detection of Salmonella colloidal gold immunochromatographic test strip in this invention are shown, wherein 1 to 12 are: Salmonella, Staphylococcus chromogenic, Staphylococcus slow-growing, Streptococcus lactis, Staphylococcus hominis, Staphylococcus aureus, Streptococcus agalactiae, Escherichia coli, Enterococcus haematobium, Streptococcus dysgalactiae, Listeria monocytogenes LM90, and Listeria monocytogenes LM-EDGe; Figure 8 The results of the sensitivity detection of the Salmonella colloidal gold immunochromatographic test strip in this invention are shown, wherein the total number of colonies 1-6 is 1×10⁻⁶. 6 1×10 5 1×10 4 1×10 3 1×10 2 1×10 1 CFU / mL. Detailed Implementation

[0017] The embodiments of the present invention will be described in detail below with reference to specific examples. The following examples are only for illustrating the present invention and should not be regarded as limiting the scope of the present invention. Rather, they should be understood as a more detailed description of certain aspects, features, and implementation schemes of the present invention.

[0018] I. Sample Source of the Invention Salmonella, Streptococcus dysgalactiae, Staphylococcus aureus, Streptococcus lactis, Staphylococcus hominis, Staphylococcus aureus, Streptococcus agalactiae, Escherichia coli, Enterococcus haematobium, LM90, LM EDG-e, and Staphylococcus chromogenicus were all stored by the Zoonotic Diseases Laboratory of Shihezi University.

[0019] II. Main reagents used in this invention The main reagent names and manufacturers are listed in Table 1.

[0020] Table 1 Main Reagents

[0021] III. Main Instruments Used in This Invention The main instrument names and manufacturers are listed in Table 2. Table 2 Main Instruments IV. Specific Implementation Examples Example 1: Preparation of Salmonella Monoclonal Antibody 1.1 Animal Immunization Six BALB / c mice approximately 6 weeks old were randomly selected. They were named pre-immunization, posterior-immunization, left-immunization, right-immunization, middle-immunization, and zero-immunization, along with one blank control. Before immunization, serum was collected from the tail vein of each mouse. The serum was allowed to separate in a 37°C incubator and then stored at -80°C. For the first immunization, Salmonella and Freund's complete adjuvant were mixed in equal proportions and administered via subcutaneous injection at multiple sites. Two weeks later, the immunogen was mixed with Freund's incomplete adjuvant and administered via intraperitoneal injection. The procedures for the third and fourth immunizations were the same as for the second immunization. The immunization dose was 50 μg per mouse.

[0023] 1.2 Antiserum titer detection About one week after the mice were immunized four times, serum was collected from the tail vein of the mice for titer testing.

[0024] (1) Dilute Salmonella to 1 μg / mL using 1×ELISA coating solution, add 100 μL / well to the microplate, and store at 4°C overnight.

[0025] (2) The next day, discard the coating solution in the well, add 200 μL of 1×PBST washing solution to each well, soak and wash three times, discard the liquid each time and pat dry, then add 200 μL of 5% BSA blocking solution to each well and place in a 37℃ incubator for 2 h to block.

[0026] (3) Discard the blocking solution, wash three times with 1×PBST and pat dry; dilute the collected mouse serum with antibody diluent at an initial concentration of 1:1000 to prepare a series of diluted serums, add 100 μL / well to the ELISA plate, and incubate in a 37℃ incubator for 1 h.

[0027] (4) Discard the serum sample in the well, wash three times with 1×PBST and pat dry, dilute the goat anti-mouse IgG-HRP enzyme-labeled secondary antibody with antibody dilution buffer at a ratio of 1:5000, add 100μL to each well into the enzyme-labeled plate, and incubate in a 37℃ incubator for 1h.

[0028] (5) Discard the enzyme-labeled secondary antibody, wash five times with 1×PBST and pat dry, add TMB single-component colorimetric solution, and react at room temperature for 20 min.

[0029] (6) Add ELISA stop solution, measure the absorbance values ​​of positive and negative wells at 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader, and record them.

[0030] The results show that ( Figure 1 The titer of mice after the fourth immunization reached 1:16000, which met the requirements for cell fusion experiments. Right mouse and no mouse were selected for fusion experiments.

[0031] 1.3 Cell Fusion After resuscitation, myeloma cells were washed, counted, and prepared into a single-cell suspension. The suspension was then resuspended in DMEM to obtain a myeloma cell resuspension. Mouse spleens were aseptically collected, and a single-splenic cell suspension was prepared using HAT medium containing 20% ​​fetal bovine serum. This spleen cell suspension was mixed with DMEM medium in an equal proportion and then added to the myeloma cell resuspension. The mixture was centrifuged, the supernatant was discarded, and the cell pellet was retained. 50% PEG 1450 was added to the cell pellet, and the reaction was carried out for 1 min. After stopping the DMEM treatment, the cells were centrifuged again, and the fused cells were collected. The cells were resuspended in HAT medium containing 20% ​​fetal bovine serum and cultured at a rate of 2 × 10⁻⁶ cells / mL. 7 The standard cells were spread into a pre-prepared 96-well plate containing feeder cells and incubated in a CO2 incubator for 8 days.

[0032] 1.4 Hybridoma cell screening and line establishment (1) Fusion screening and subcloning detection Once the hybridoma cells in the 96-well plates were in good condition, the following procedures were performed: Salmonella was diluted to 1 μg / mL with 1×PBS and coated with ELISA solution, then incubated overnight at 4°C. The next day, 100 μL of cell supernatant was aseptically aspirated from each well for ELISA detection. Positive values ​​were screened for subcloning. Liquid from wells with high positive values ​​was picked and subjected to limiting dilution. Titer was measured 7 days after each subcloning. After multiple subcloning and detection cycles, a monoclonal cell line that stably secreted positive antibodies was finally selected.

[0033] (2) Cell line establishment Cell lines selected in the previous stage were expanded and cultured in 24-well plates. The supernatant was collected for titer testing. After the titer was qualified, the cells were transferred to 10cm culture dishes for further expansion and culture. The cell supernatant was collected again and the antibody titer was detected by ELISA. Cell lines with high titers were selected and transferred to T25 cell culture flasks for further culture. Antibody subtype detection kits were used to identify the antibody subtypes of the cell lines.

[0034] Screening results: A total of 5 cell lines with high cell titers were screened (all were monoclonal cell lines purified by multiple subcloning processes), and were named 7B9 2D2, 2F12 1D12, 9B12 2E5, 4H10 3F1, and 8E7 1C8, respectively. The antibody subtype identification of the 5 cell lines is shown in Table 3.

[0035] Table 3. Identification of antibody subtypes in cell lines

[0036] 1.5 Preparation and purification of monoclonal antibodies Antibodies were prepared from the 8E7 1C8 cell line, which had higher titers, using ELISA.

[0037] (1) Preparation of ascites Four-week-old mice were pretreated with liquid paraffin via intraperitoneal injection 14 days in advance; the density of expanded 8E7 1C8 monoclonal hybridoma cells was increased to 1×10⁻⁶. 6 ~2×10 6 / mL, after centrifugation, discard the supernatant, resuspend the cells in 1mL DMEM, inject intraperitoneally into pretreated mice, collect the ascites fluid, and store at -20℃.

[0038] (2) Antibody purification 1) The collected ascites fluid was treated with 50% SAS precipitation method, the precipitated sample was collected and transferred to phosphate buffer for overnight dialysis; after dialysis, it was filtered and sterilized using a 0.22μm filter membrane and kept for later use.

[0039] 2) Column equilibration: Wash the Protein G purification column with deionized water to remove the ethanol, and then elute with 0.01M PBS to equilibrate the purification column.

[0040] 3) Load the filtered and sterilized sample onto a pre-equilibrated Protein G purification column. Use 0.1 mmol / L glycine hydrochloric acid (pH 2.7) as the elution buffer to elute the target antibody. Collect the target eluent and add 1 mol / L Tris-HCl (pH 9.0) to the target eluent for neutralization. Store the neutralized purified antibody at -20°C for later use.

[0041] (3) Monoclonal antibody titer detection 1) Dilute Salmonella to 1 μg / mL using 1×ELISA coating buffer, add it to the ELISA plate, and bind.

[0042] 2) The next day, discard the waste liquid and elute three times with PBST, 5 min each time. Add skim milk powder to block the reaction and incubate at 37°C for 2 h.

[0043] 3) Discard the blocking solution, repeat the previous steps to wash, pat dry, serially dilute the purified Salmonella monoclonal antibody 8E7 1C8 (hereinafter referred to as 8E7 1C8 monoclonal antibody), add it to the ELISA plate, and react at 37°C for 1 hour.

[0044] 4) Discard the monoclonal antibody, repeat the previous steps to wash, pat dry, add 100 μL of goat anti-mouse enzyme-labeled secondary antibody (1:5000 dilution) to each well, and react at 37℃ for 1 h.

[0045] 5) Discard the enzyme-labeled antibody, repeat the previous steps to wash, pat dry, add single-component TMB colorimetric solution, and react at room temperature for 20 minutes.

[0046] 6) Add ELISA stop solution, measure the OD value at 450nm absorbance, and record it.

[0047] The results show that ( Figure 2 Even at a concentration as low as 0.015625 μg / mL, the purified 8E7 1C8 monoclonal antibody showed an OD value 2.1 times higher than the negative control value in its reaction with Salmonella antigen, indicating that the monoclonal antibody has high affinity.

[0048] (4) Monoclonal antibody purity detection Antibody purity was determined using SDS-PAGE.

[0049] 1) Prepare a 12% separating gel and slowly add it to a glass plate. After it solidifies, prepare a 5% concentrating gel and insert a gel comb to allow it to solidify naturally.

[0050] 2) Add freshly prepared electrophoresis buffer to the electrophoresis tank. Dilute the purified 8E7 1C8 monoclonal antibody 4-fold with sample buffer and slowly add 5 μL of sample to the sample well. First, perform electrophoresis at 80V and 300mA. After the bands have completely emerged from the stacking gel, switch to 120V and 300mA and continue electrophoresis until the indicator reaches the bottom of the gel block.

[0051] 3) Turn off the electrophoresis apparatus, gently remove the separating gel, place it in a glass dish, add Coomassie Brilliant Blue staining solution and stain for 10 minutes. Then, discard the staining solution and add destaining solution to decolorize the gel block until it becomes transparent.

[0052] 4) Take pictures and analyze them using a luminescence analyzer.

[0053] The results show that ( Figure 3 The purified 8E7 1C8 monoclonal antibody showed only single heavy and light chain bands, indicating high purity. Furthermore, the darker color of the bands indicated a higher concentration.

[0054] (5) Monoclonal antibody sequencing To obtain the precise variable region sequence of the 8E7 1C8 monoclonal antibody, hybridoma cell lines were sent to General Biotechnology (Anhui) Co., Ltd. for sequencing. The company extracted total RNA from the cells, synthesized cDNA via reverse transcription, and performed PCR amplification using mouse IgG antibody variable region-specific primers to obtain heavy chain variable region (VH) and light chain variable region (VL) gene fragments, respectively. After cloning and sequencing, the coding sequences are as follows: The base sequence encoding the variable region of the heavy chain (SEQ ID NO:1): ggaggcttggtacagcctgggggttctctgagactctcctgtgcaacttctgggttcaccttcactgattactacatgagctgggtccgccagcctccaggaaaggcacttgagtggttgggttttattagaaacaaagctaatggttacacaacagagtacagtgcatctgtgaagggtcggttcaccatctccagagataattcccaaagcatcctctatcttcaaatgaacaccctgagagctgaggacagtgccacttattactgtgcaagacccctctatggtaactactggtacttcgatgtctggggcgcaggg; Heavy chain variable region amino acid sequence (SEQ ID NO: 2): GGLVQPGGSLRLSCATS GFTFTDYY MSWVRQPPGKALEWLGF IRNKANGYTT EYSASVKGRFTISRDNSQSILYLQMNTLRAEDSATYYC ARPLYGNYWYFDV WGAG; Base sequence encoding the light chain variable region (SEQ ID NO: 3): atggagacagagacactcctgctatgggtgctactgctctgggttccaggttccacaggtaaaattgtgctgacccaatctccagcttctttggctgtgtctctaagacagagggccaccatatcctgcagagccagtgaaagtgttgatagttatggcaatagttttatgcactggtaccagcagaaaccaggacagccacccaaactcctcatctatcgtgcatccaagctagaatctggggtccctgccaggttcagtggcagtgggtctaggacagacttcaccctcaccattgatcctgtggaggctgatgatgctgcaacctattactgtcagcaaaataatgaggatccgtggacgttcggtggaggcactaagctggaaatcaaacgg; Light chain variable region amino acid sequence (SEQ ID NO: 4): METETLLLWVLLLWVPGSTGKIVLTQSPASLAVSLRQRATISCRASE SVDSYGNSF MHWYQQKPGQPPKLLIY RAS KLESGVPARFSGSGSRTDFTLTIDPVEADDAATYYC QQNNEDPWT FGGGTKLEIKR.

[0055] Sequence analysis based on the IMGT database identified the amino acid sequences of the three heavy chain variable regions complementarity-determining regions of this monoclonal antibody as follows: VH-CDR1 (SEQ ID NO 5): GFTFTDYY; VH-CDR2 (SEQ ID NO 6):IRNKANGYTT; VH-CDR3 (SEQ ID NO 7): ARPLYGNYWYFDV; The specific locations of the three complementarity-determining regions are the regions shown underlined in the heavy chain variable region amino acid sequence (SEQ ID NO:2).

[0056] The amino acid sequences of the complementarity-determining regions of the three light chain variable regions are as follows: VL-CDR1 (SEQ ID NO 8):SVDSYGNSF; VL-CDR2:RAS; VL-CDR3 (SEQ ID NO 9): QQNNEDPWT.

[0057] The specific locations of the three complementarity-determining regions are the regions shown underlined in the light chain variable region amino acid sequence (SEQ ID NO:4).

[0058] Example 2: Preparation of Salmonella colloidal gold immunochromatographic test strip based on monoclonal antibody 2.1 Preparation and purification of Salmonella polyclonal antibodies 2.1.1 Preparation of Salmonella polyclonal antibodies Pure Salmonella cultures were cultured in BHI medium for 14 hours until the OD value was between 0.4 and 0.6. The total bacterial count was determined using a turbidimeter, and 1 mL of the bacterial suspension was sonicated at 200 W.

[0059] One week prior to immunization, negative serum was collected from the ear vein of the rabbits. The whole bacterial antigen was emulsified with 1 mL of Freund's complete adjuvant, and the initial immunization was performed subcutaneously at five points on the back. Repeat immunizations were given every 7 days (immunization protocol as shown in Table 4). Blood was collected periodically during the immunization process, and titers were determined using indirect ELISA. Once the serum met the required standards, blood was drawn from the heart of the rabbits, and the serum was collected.

[0060] Table 4. Salmonella polyclonal antibody immunization regimen

[0061] 2.1.2 Purification of Salmonella polyclonal antibodies (1) Crude antibody extraction by saturated ammonium sulfate precipitation method Centrifuge 10 mL of serum at 12000 rpm for 0.5 h. Add an equal volume of saturated ammonium sulfate to the supernatant, stir with a magnetic stirrer for 4 hours, and then incubate at 4°C for 12 h. Add 6 mL of 1×PBS and 4 mL of saturated ammonium sulfate solution to the precipitate after a second centrifugation, mix well, and incubate at 4°C for 1 h. Transfer the solution to a dialysis bag and perform dialysis in PBS.

[0062] (2) Antibody purification using affinity chromatography column After crude purification, the sample was added to an antibody purification column for further purification. The concentration of the purified antibody was then determined using a Nano Drop 2000.

[0063] 2.2 Prepare 8E7 1C8 monoclonal antibody according to the method in Example 1 2.3 Optimization of conditions for colloidal gold-labeled monoclonal antibodies (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 30 nm colloidal gold solution was added to each well. Well 1 served as the control group and received no treatment. The pH of the colloidal gold solution in wells 2-10 was adjusted using 0.1 mol / L 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. 10 μL of 1 mg / mL 8E7 1C8 monoclonal antibody was added to each well, and 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 incubation at room temperature for 2 h. The color change of the colloidal gold solution was observed.

[0064] The results show that ( Figure 4 When the pH of the colloidal gold solution is 6, the colloidal gold solution binds best to the 8E7 1C8 monoclonal antibody, with no obvious color change. Therefore, the optimal pH for the binding of the 8E7 1C8 monoclonal antibody to colloidal gold is 6.

[0065] (2) Exploration of the optimal concentration of colloidal gold-labeled monoclonal antibody Take eight sample wells from a removable 96-well ELISA plate and add 0.3 mL of 30 nm colloidal gold solution to each well. After adjusting the colloidal gold solution to the optimal pH value, add 0 μL, 0.6 μL, 1.2 μL, 1.8 μL, 2.4 μL, 3.0 μL, 4.5 μL, and 6.0 μL of 8E7 1C8 monoclonal antibody, respectively, to achieve final 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. Mix well and incubate at 37℃ for 1 h. Add 30 μL of 10% NaCl solution and let stand at room temperature for 1 h. The optimal binding condition between the monoclonal antibody and the colloidal gold particles is when there is no color change in the colloidal gold sol.

[0066] The results show that ( Figure 5 When the labeling amount of 8E7 1C8 monoclonal antibody was 6 μg / mL, there was no obvious visual change in the labeled colloidal gold solution. Based on a comprehensive consideration of stability and signal intensity, 6 × 1.2 = 7.2 μg / mL was selected as the optimal coating amount for colloidal gold-labeled Salmonella monoclonal antibody.

[0067] 2.4 Pretreatment of the sample pad The sample pad is treated with a buffer solution to improve the binding and release of the sample to be tested and the gold-labeled antibody on the gold-labeled pad. Pretreatment with a mixture of 0.2% Tween-20 and 3% sucrose is preferred.

[0068] Pretreatment of 2.5 gold standard pad The gold-labeled pads were soaked in a 2% sucrose solution for 20 minutes and then dried in an incubator at 37°C.

[0069] 2.6 Selection of Optimal Coating Concentration for T-Line Antibodies Take 10 mL of colloidal gold solution, adjust to the optimal pH value, slowly add 72 μL of 1 mg / mL 8E7 1C8 monoclonal antibody, add 10% BSA for blocking after reaction, and then use it after resuspension and reconstitution.

[0070] The Salmonella polyclonal antibody prepared in Example 2.1 was diluted to 2.0 mg / mL, 1.5 mg / mL, 1.0 mg / mL, and 0.5 mg / mL, respectively, and used as T-line streaking solutions. The C-line was prepared using goat anti-mouse IgG (purified antibody) at a concentration of 1.0 mg / mL as the secondary antibody, and the test strips were assembled.

[0071] The results show that ( Figure 6 When the concentration of the T line was 2 mg / mL, the colorimetric results were significantly better than those of other groups.

[0072] 2.7 Salmonella Monoclonal Antibody Test Strip Assembly (1) Preparation of colloidal gold solution labeled with 8E7 1C8 monoclonal antibody: Take a clean glass bottle, add a certain amount of colloidal gold solution, and slowly add 0.1 mol / L K2CO3 while stirring to adjust the pH of the colloidal gold solution to 6. Add 7.2 μg / mL of 8E7 1C8 monoclonal antibody, and incubate at 37℃ for 1 h. Add 10% BSA and continue the reaction for 0.5 h. Transfer the solution to a new centrifuge tube, centrifuge at 12000 rpm for 10 min, discard the supernatant, resuspend the precipitate with resuspension solution and centrifuge again. Repeat three times. Finally, resuspend the precipitate with colloidal gold resuspension solution and store at 4℃ for later use. (2) Immerse the pretreated gold pad in the above colloidal gold solution for 30 minutes, and let it air dry naturally for later use; (3) Using goat anti-mouse IgG (purified antibody) at a concentration of 1.0 mg / mL as the C line and Salmonella polyclonal antibody at a concentration of 2.0 mg / mL as the T line, the C line and T line were marked on the NC membrane using a membrane scrubbing instrument; (4) Cut the sample pad to 1.5cm wide, the gold label pad to 0.8cm wide, and the absorbent pad to 1.8cm wide. Attach the NC film, gold label pad, sample pad, and absorbent pad to the PVC base plate in sequence. Cut into strips using a chopper.

[0073] Example 3: Performance testing of Salmonella colloidal gold immunochromatographic test strips The Salmonella colloidal gold immunochromatographic test strips prepared by the above optimized method were subjected to the following specificity and sensitivity tests.

[0074] 3.1 Specificity detection of Salmonella using colloidal gold immunochromatographic test strips Samples of laboratory-preserved bacteria—Salmonella, Staphylococcus chromogenic, Staphylococcus slow-growing, Streptococcus lactis, Staphylococcus hominis, Staphylococcus aureus, Streptococcus agalactiae, Escherichia coli, Enterococcus haematobium, Streptococcus dysgalactiae, Listeria monocytogenes LM90, and Listeria monocytogenes LM-EDGe—were added to the test strips for specific detection.

[0075] The results show that ( Figure 7 The established colloidal gold test strip showed no cross-reactivity with other bacteria, indicating that the test strip has good specificity.

[0076] 3.2 Sensitivity detection of Salmonella colloidal gold immunochromatographic test strips After determining the total colony count using a turbidimeter, the activated bacterial strain was serially diluted to 1×10⁻⁶ using PBS. 6 1×10 5 1×10 4 1×103 1×10 2 1×10 1 CFU / mL was added to the assembled test strips, and the color development of the T and C lines on the test strips was observed and recorded.

[0077] The results show that ( Figure 8 The colloidal gold immunochromatographic test strip of the present invention has a total bacterial count of 1×10⁻⁶. 3 1×10 2 At CFU / mL, a blurred T line is visible; when the total bacterial count detected by the test strip is 1×10⁻⁶, a faint T line is observed. 4 At CFU / mL, the T line is clearly visible, therefore the sensitivity of the test strip is approximately 1×10⁻⁶. 4 CFU / mL.

[0078] 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 Salmonella monoclonal antibody, characterized in that, The monoclonal antibody is named 8E7 1C8, and its heavy chain variable region includes three complementarity-determining regions: VH-CDR1, VH-CDR2, and VH-CDR3, wherein: The amino acid sequence of VH-CDR1 is GFTFTDYY; The amino acid sequence of VH-CDR2 is IRNKANGYTT; The amino acid sequence of VH-CDR3 is ARPLYGNYWYFDV; The light chain variable region of the monoclonal antibody 8E7 1C8 includes three complementarity-determining regions: VL-CDR1, VL-CDR2, and VL-CDR3, wherein: The amino acid sequence of VL-CDR1 is SVDSYGNSF; The amino acid sequence of VL-CDR2 is RAS; The amino acid sequence of VL-CDR3 is QQNNEDPWT.

2. The Salmonella monoclonal antibody as described in claim 1, characterized in that, The amino acid sequence of the heavy chain variable region of the monoclonal antibody 8E7 1C8 is SEQ ID NO:

2.

3. The Salmonella monoclonal antibody as described in claim 1, characterized in that, The amino acid sequence of the light chain variable region of the monoclonal antibody 8E7 1C8 is SEQ ID NO:

4.

4. The Salmonella monoclonal antibody according to any one of claims 1 to 3, characterized in that, The heavy chain constant region of the monoclonal antibody 8E71C8 is of type IgG2b.

5. The use of the Salmonella monoclonal antibody as described in claim 4 in the preparation of Salmonella detection products.

6. A reagent or kit for detecting Salmonella, characterized in that, It contains the Salmonella monoclonal antibody as described in claim 4.

7. A colloidal gold immunochromatographic test strip for detecting Salmonella, 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 Salmonella monoclonal antibody as described in claim 4.

8. The colloidal gold immunochromatographic test strip as described in claim 7, characterized in that, The coating concentration of the Salmonella monoclonal antibody was 7.2 μg / mL.

9. The colloidal gold immunochromatographic test strip as described in claim 7, characterized in that, The detection line is coated with Salmonella polyclonal antibody at a concentration of 2 mg / mL.

10. A method for preparing the Salmonella monoclonal antibody according to claim 4, characterized in that, Includes the following steps: (a) Immunization procedure: BALB / c mice were immunized with Salmonella as an immunogen at a dose of 50 μg / mouse. (b) Screening before fusion: Mice with serum titers ≥1:16000 were screened by indirect ELISA after immunization, and their spleen cells were fused with myeloma cells mediated by a chemical fusion agent. (c) Cell screening and line establishment: Salmonella was used as an antigen to screen positive hybridoma cells. After subcloning, cell lines with heavy chain constant region of IgG2b type and CDR region sequence of secreting antibody conforming to SEQ ID NO:5~10 were screened to obtain monoclonal hybridoma cell lines that can stably secrete the monoclonal antibody 8E7 1C8. (d) Antibody acquisition and quality control: The above cell lines were cultured, and antibodies were produced by ascites fluid preparation or cell culture. After affinity chromatography purification, the specific binding and titer of the antibodies to Salmonella were verified.

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