Colloidal gold immunochromatography test strip for listeria monocytogenes as well as preparation method and application of colloidal gold immunochromatography test strip

Abstract

The invention belongs to the technical field of colloidal gold immunochromatography, and particularly relates to a colloidal gold immunochromatography test strip for listeria monocytogenes as well as a preparation method and application of the colloidal gold immunochromatography test strip for the listeria monocytogenes, the test strip comprises a bottom plate, and a sample pad, a gold label pad, a detection pad and a water absorption pad which are sequentially arranged on the bottom plate, the concentration of the monoclonal antibody of the listeria monocytogenes in the colloidal gold pad is 4-30 [mu] g / mL, and the prepared colloidal gold test strip has good specificity and does not generate cross reaction with other pathogenic bacteria in cow milk.

Description

Technical Field

[0001] This invention relates to the field of colloidal gold immunochromatography technology, specifically to a colloidal gold immunochromatographic test strip for Listeria monocytogenes, its preparation method, and its application. Background Technology

[0002] Milstein and Köhler developed a method for producing large quantities of single, custom-specific monoclonal antibodies by fusing mouse myeloma cell lines with mouse spleen B lymphocytes to create hybridoma cells. Hybridoma cells are cell lines that can grow in culture and produce immunoglobulins with the same amino acid sequence, namely monoclonal antibodies (mAbs), thus exhibiting high affinity and specificity for a single epitope of an antigen. Hybridoma cells not only retain the antibody-producing ability of B lymphocytes but also, like hybridoma cells, can proliferate continuously. Monoclonal antibodies bind to antigens through specific regions they possess. This specificity of monoclonal antibodies reduces the probability of off-target effects, and many monoclonal antibodies have very high affinity, typically less than 1 nM. Monoclonal antibodies can be produced targeting any given epitope present on an antigen or immunogen, and with the maturation of monoclonal antibody production technologies and the optimization of production methods, they have become a powerful tool in the fields of biochemistry, molecular biology, and medicine.

[0003] Colloidal gold immunochromatographic assay (GICA) is an immunological detection method that uses a cellulose membrane as a carrier and colloidal gold particles-labeled antigens or antibodies as tracers. Due to its advantages of simple operation, convenience, speed, and intuitive results, GICA is widely used in the detection of pathogenic microorganisms, pesticides, antibiotics, food additives, and biomarkers.

[0004] Listeria monocytogenes is an important foodborne pathogen. When using colloidal gold immunochromatography to detect it, two types of antibodies are mainly used: anti-internalin A (InlA) monoclonal antibody and anti-internalin B (InlB) polyclonal antibody. However, in actual samples, it is still difficult to completely get rid of cross-linking and background adsorption, resulting in low specificity. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a colloidal gold immunochromatographic test strip for Listeria monocytogenes, its preparation method, and its application.

[0006] A colloidal gold immunochromatographic test strip for Listeria monocytogenes, the test strip comprising a base plate and a sample pad, a gold label pad, a detection pad and an absorbent pad sequentially disposed on the base plate, wherein the concentration of the monoclonal antibody of Listeria monocytogenes in the gold label pad is 4-30 μg / mL; The monoclonal antibody against Listeria monocytogenes is 1H6 1B8. The nucleotide sequence of the heavy chain variable region of 1H6 1B8 is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2. The nucleotide sequence of the light chain variable region of 1H6 1B8 is shown in SEQ ID NO.3, and the amino acid sequence is shown in SEQ ID NO.4.

[0007] When using the monoclonal antibody against Listeria monocytogenes provided by this invention to detect Listeria monocytogenes in colloidal gold immunochromatographic test strips, when Listeria monocytogenes is present in the sample, it will be captured by the gold-labeled Listeria monocytogenes monoclonal antibody on the gold-labeled pad. The prepared colloidal gold test strip has good specificity and does not cross-react with other pathogens in milk.

[0008] Preferably, the pH of the gold-labeled pad is 6-9.5.

[0009] Preferably, the amount of antibody coated on the detection line of the detection pad is 0.5-2.0 mg / mL.

[0010] The preparation method of the colloidal gold immunochromatographic test strip includes the following steps: Under conditions of pH 6-9.5, colloidal gold and monoclonal antibody against Listeria monocytogenes were added and incubated. The ratio of colloidal gold to monoclonal antibody against Listeria monocytogenes was 10 nM: 2 μg / mL~30 μg / mL. Set the polyclonal antibody against Listeria monocytogenes as the detection line; Assembly of colloidal gold test strips.

[0011] Preferably, the method for preparing the monoclonal antibody against Listeria monocytogenes includes the following steps: Mice were immunized with a mixture of inactivated Listeria monocytogenes and Freund's adjuvant. Spleen cells and myeloma cells from immunized mice were mixed at a ratio of 5-10:1 to obtain hybridoma cells through cell fusion. Positive hybridoma cells capable of producing Listeria monoclonal antibodies were inoculated into the peritoneal cavity of mice, and the ascites fluid was collected and purified to obtain the monoclonal antibodies against Listeria monoclonal.

[0012] Preferably, the assembly of the colloidal gold test strip includes the following steps: Place the gold-labeled pad in colloidal gold-labeled monoclonal antibody and soak it for later use; Mark the test lines and control lines on the nitrocellulose membrane; The sample pad, gold label pad, and nitrocellulose membrane were sequentially placed on a polyethylene chloride substrate.

[0013] The application of the colloidal gold immunochromatographic test strip in the detection of Listeria monocytogenes.

[0014] Preferably, the test solution is loaded onto the colloidal gold immunochromatographic test strip, and the results are observed and determined after standing for 8 to 12 minutes.

[0015] Preferably, the criteria for judgment are: (a) Negative: If only the control line (C) shows a visible band, while the test line (T) shows no visible band, the test result is negative, indicating that the sample does not contain Listeria monocytogenes or its content is below the detection limit. (b) Positive: If visible bands appear on both the test line (T) and the control line (C), the test result is positive, indicating that the sample contains Listeria monocytogenes. (c) Invalid: If no visible band appears on the control line (C), the test is considered invalid regardless of whether a band appears on the test line (T). Invalid results indicate that the test strip may be invalid or the procedure may be incorrect; it is recommended to retest with a new test strip.

[0016] This invention uses Listeria monocytogenes as an immunogen. Following an immunization program involving mouse immunization, cell fusion, and subcloning, four hybridoma cell lines capable of stably secreting monoclonal antibodies were ultimately screened. These four cell lines were then identified as having IgG1 and IgG2b subtypes. IgG is a tetramer composed of two identical heavy chains (HC) (50 kDa) and two identical light chains (LC) (25 kDa) linked by interchain disulfide bonds. Based on minor differences in the constant HC region, IgG is further divided into four subclasses (IgG1, IgG2, IgG3, and IgG4). The main difference lies in the hinge region connecting the two identical HC / LC pairs; the hinge length and disulfide bond pattern differ among the different subclasses. The monoclonal antibodies prepared using mice as a model are mainly of the following subtypes: IgG1, IgG2a, and IgG2b. The monoclonal antibody subtypes screened in this invention are common subtypes. This may be related to the higher conformational stability of the IgG1 subtype. The screening of the IgG2 subtype may be related to the pH during screening, which is attributed to the fact that the IgG2b subtype is a pH-sensitive IgG.

[0017] Colloidal gold solutions exhibit strong intrinsic stability within a pH range of 5.0-9.0. When antibodies are added, they disrupt the electrostatic charge on the surface of the colloidal gold particles, causing self-copolymerization and altering the color. Therefore, adjusting the pH of the antibody-colloidal gold mixture can reduce self-copolymerization and increase the stability of antibody-colloidal gold binding, resulting in clear and distinct red lines on both the detection and control lines. Furthermore, the binding of colloidal gold to antibodies is related to the diameter of the colloidal gold particles, the concentration of the colloidal gold solution, the relative molecular mass of the antibody, and the isoelectric point of the protein. In this invention, the optimal labeling pH for antibody-labeled colloidal gold particles is 6.0, and the optimal labeling concentration is 4 μg / mL.

[0018] This invention utilizes a polyclonal antibody-coated detection line against Listeria monocytogenes. Colloidal gold particles are labeled with a prepared monoclonal antibody against Listeria monocytogenes. When Listeria monocytogenes is present in the sample, it is captured by the gold-labeled Listeria monocytogenes monoclonal antibody on the gold-labeled pad. As the liquid flows to the detection line, Listeria monocytogenes is further captured by the polyclonal antibody against Listeria monocytogenes. As the amount of captured product increases, a visible red line appears on the detection line. Therefore, the amount of antibody coating on the detection line directly affects the sensitivity of the colloidal gold test strip, thus requiring optimization. The results of this invention show that the detection line is clear when the antibody coating concentration is 2.0 mg / mL. When the specificity and sensitivity of the colloidal gold test strip prepared under optimal conditions were evaluated, the results showed that the colloidal gold test strip prepared in this invention has good specificity and does not cross-react with other pathogens in milk. This can be attributed to the use of monoclonal antibodies, which to some extent increases the accuracy of the detection line in capturing the target strain. Furthermore, the colloidal gold immunochromatographic detection line established in this study is 1×10⁻⁶. 5 CFU / mL.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention uses the high-titer cell line 1H6 1B8 for the preparation and purification of monoclonal antibodies. The purified antibody titer reached 1:192000. SDS-PAGE verification analysis showed that the purified monoclonal antibody had no impurities and the bands were clear. Its heavy chain size was approximately 55 kDa, and its light chain size was approximately 25 kDa, which is consistent with the composition (heavy and light chains) of IgG antibodies. Using a polyclonal antibody-coated detection strip against Listeria monocytogenes, colloidal gold particles were labeled with the prepared anti-Listeria monocytogenes monoclonal antibody. When Listeria monocytogenes was present in the sample, it was captured by the gold-labeled Listeria monocytogenes monoclonal antibody on the gold-labeled pad. The prepared colloidal gold test strip exhibited good specificity and did not cross-react with other pathogens in milk. Attached Figure Description

[0020] Figure 1 This is a schematic diagram of the structure of a colloidal gold test strip.

[0021] Figure 2 For the detection of monoclonal antibody titers.

[0022] Figure 3 The results are for SDS-PAGE validation of the monoclonal antibody. M: protein marker; 1: 1H6 1B8 monoclonal antibody.

[0023] Figure 4 To determine the optimal pH for monoclonal antibody labeling of colloidal gold, 1: stock solution (initial pH: 5.0); 2-10: pH values ​​were 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, and 9.5, respectively.

[0024] Figure 5 To determine the optimal labeling concentration of monoclonal antibodies, 1: no antibody added, i.e., 0 μg / mL; 2-9: antibody labeling concentrations of 2 μg / mL, 4 μg / mL, 6 μg / mL, 8 μg / mL, 10 μg / mL, 15 μg / mL, 20 μg / mL, and 30 μg / mL, respectively.

[0025] Figure 6 To determine the optimal coating amount for the detection line, the antibody coating concentrations for steps 1-4 are 2 mg / mL, 1.5 mg / mL, 1 mg / mL, and 0.5 mg / mL, respectively.

[0026] Figure 7 For specificity of colloidal gold immunochromatography, 1: Listeria monocytogenes LM90 2-10 are Staphylococcus aureus, Streptococcus agalactiae, Escherichia coli, Enterococcus haematobium, Streptococcus galactiae, Salmonella, Staphylococcus chromogenic, Staphylococcus stenotic, and Streptococcus lactis, respectively; 11: negative control.

[0027] Figure 8 To improve the sensitivity of colloidal gold immunochromatography, 1-6: 1×10 7 CFU / mL - 1×10 2 CFU / mL Listeria monocytogenes. Detailed Implementation

[0028] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention. Unless otherwise specified, the experimental methods described in the embodiments of the present invention are conventional methods.

[0029] The strain used in this invention is the same as in Experiment 1. The myeloma cells Sp2 / 0 were preserved by the Zoonotic Diseases Laboratory of the College of Animal Science and Technology, Shihezi University.

[0030] The results for the heavy chain variable region of 1H6 1B8 are as follows: Base sequence: caggtgcagctgaagcagtcaggagctggcctagtgcagccctcacagagcctgtccatcacctgcacagtctctggtttctcattaactaaccatggtgtacactgggttcgccagtctccaggaaagggtctggagtggctgggagtgatatggagtggtggacgcacagact atagtgcagctttcatatccagactgagcatcagcaaggacaattccaagaggcaagctttctttaaaatggacagtctgcaagctaatgacacagccatatattactgtgccagtgatttctcctatgctatggactactggggtcaaggaacctcagtcaccgtctcctcag, denoted as SEQ IDNO.1.

[0031] Amino acid sequence: QVQLKQSGAGLVQPSQSLSITCTVSGFSLTNHGVHWVRQSPGKGLEWLGVIWSGGRTDYSAAFISRLSISKDNSKRQAFFKMDSLQANDTAIYYCASDFSYAMDYWGQGTSVTVSS, recorded as SEQ ID NO. 2.

[0032] The results for the light chain variable region of 1H6 1B8 are as follows: Base sequence: gacatccagctgactcagtctccagcctccctatatgcatctgtgggagaaactgtcaccatcacatgtcgagtaagtgagaatatttacaggtatttagcatggtatcagcagaaacagggaaaatctcctcagttcctggtctacaatgcaaaaacgtt agtagaaggtgtgccatcaaggttcagtggcagtggatcaggcacacagttttctctgaagatcaacaccctgcagcctgaagattttgggagttattattgtcaaaatcattatgggactcctctcgcgttcggtgctgggaccaagttggagctgaaac, denoted as SEQ ID NO.3.

[0033] Amino acid sequence: DIQLTQSPASLYASVGETVTITCRVSENIYRYLAWYQQKQGKSPQFLVYNAKTLVEGVPSRFSGSGSGTQFSLKINTLQPEDFGSYYCQNHYGTPLAFGAGTKLELK, denoted as SEQ ID NO.4.

[0034] Example 1 I. Preparation of Monoclonal Antibodies 1. Animal immunization (1) Preparation of immunogen: Listeria monocytogenes were cultured overnight until the OD value was 0.6, inactivated in a water bath at 80℃ for 30 min, collected by centrifugation and resuspended in physiological saline to obtain a bacterial suspension for later use; (2) Animal selection: Ten 6-8 week old female BALB / c mice were randomly divided into two groups, namely the experimental group and the control group. The control group was injected with PBS. (3) First immunization: Take the bacterial suspension in (1) and mix it with Freund's adjuvant in equal volume. After emulsification and mixing, the experimental group mice were immunized by intraperitoneal injection at a dose of 50 µg / mouse. (4) Second immunization: The second immunization is carried out 14 days after the first immunization. The immunogen is mixed with Freund's incomplete adjuvant at a ratio of 1:1 and immunized after complete emulsification. (5) Third immunization: The third immunization, i.e., the booster immunization, is performed 12 days after the second immunization. The immunization dose is the same as that of the first immunization. (6) Potency test: One week after booster immunization, blood was collected from the orbital vein of mice, whole blood was collected and placed in a 37°C constant temperature incubator to separate serum, and serum potency was detected by indirect ELISA; (7) Re-enzyme booster: One week later, select mice with serum titers that meet the requirements for a second booster immunization. The immunization dose is the same as the third immunization. Cell fusion is performed three days later.

[0035] 2. Serum titer detection and screening (1) Dilute Listeria monocytogenes in ELISA coating solution to 1 μg / mL, vortex to mix, and then coat 100 μL in each well of a 96-well plate. Incubate overnight at 4°C. (2) Blocking: Discard the coating solution, add 1×PBST to wash the plate 3-4 times, 5 min each time; after the last wash, pat dry the residual liquid in the wells and add blocking solution (5% BSA), 200 μL per well, and block at 37℃ for 2 h. (3) Incubation of primary antibody: After blocking, discard the blocking solution, add 200 μL of 1×PBST to each well for washing, 5 min each time, 3 times; after the last wash, pat dry the residual liquid in the well, and continuously dilute mouse serum at a ratio of 1:1000, add 100 μL to each well, and incubate at 37℃ for 1 h. (4) Incubation of secondary antibody: After the primary antibody incubation is completed, discard the primary antibody in the well, add 200 μL of 1×PBST to each well to wash the plate for 5 min each time, and wash 3 times; after the last wash, pat the residual liquid in the well dry and add 1×PBST diluted goat anti-mouse-IgG / HRP (dilution factor 1:5000), and incubate at 37℃ for 30 min. (5) Color development: After the secondary antibody incubation is completed, discard the secondary antibody in the well, add 200 μL of 1×PBST to each well to wash the plate for 5 min each time, and wash 3 times; after the last wash, pat the residual liquid in the well dry and add TMB single-component color development solution, and incubate at 37°C in the dark for 15 min. (6) Termination: Add 50 μL of ELISA termination solution to each well and measure the OD value at 450 nm. Calculate the P / N value (positive serum OD value / negative serum OD value) and use a P / N value ≥ 2.1 as the cutoff value.

[0036] 3. Cell fusion (1) Preparation of myeloma cells Frozen Sp2 / 0 myeloma cells were removed from liquid nitrogen, thawed in a water bath at 37°C, and the cell suspension was collected. The cells were centrifuged at 1000 rpm for 5 min, the supernatant was discarded, and the cells were resuspended in incomplete DMEM medium. After centrifugation and discarding the supernatant, the cells were resuspended in complete medium (FBS+DMEM) and gently pipetted. The cells were then transferred to a cell culture dish and cultured in a 37°C 5% CO2 incubator. Cell status was observed, and the medium was changed and passaged. The cells were kept in the logarithmic growth phase before confluence.

[0037] (2) Spleen cell preparation Select mice with acceptable serum titers, remove the eyeballs to collect blood, and euthanize the mice. Disinfect the mice by immersing them in 75% alcohol for 10 minutes. Place the mice in a laminar flow hood, and use autoclaved surgical scissors to cut open the abdomen, exposing the spleen. Remove the spleen with sterile forceps and place it in a 1.5 mL EP tube. Remove the fat and connective tissue from the spleen surface, wash with DMEM, gently rupture the spleen, place it in the center of a cell strainer, and while grinding with forceps, gently rinse and disperse the cells with DMEM. Centrifuge at 1000 rpm for 5 minutes, discard the supernatant, and resuspend the cells in DMEM to prepare a spleen cell suspension, ensuring a spleen cell count of 10-1. 8 It can be used for cell fusion.

[0038] (3) Preparation of feeder cells Unimmunized healthy BALB / c mice were prepared with spleen cell suspension as described in (2), and then plated in 96-well cell plates, 100 μL per well, and cultured in a 37°C incubator containing 5% CO2, and the contamination was observed.

[0039] (4) Cell fusion Myeloma cells and spleen cells were mixed in centrifuge tubes at a ratio of 1:5 to 1:10. In this example, the ratio was 1:8. The remaining experimental procedures were performed according to Shi Yiheng, Study on Preparation and Immunodetection Methods of Monoclonal Antibody of Bacillus Cyclocarya [D]. Yangling: Northwest A&F University, 2021. Finally, the 96-well cell culture plate was placed in a 37°C incubator containing 5% CO2 to obtain hybridoma cells.

[0040] 4. Preparation of hybridoma cell lines (1) Fusion screening Due to the characteristics of HAT selective medium, only hybridoma cells produced by the fusion of spleen cells and myeloma cells can grow in HAT selective medium. Unfused spleen cells or myeloma cells cannot survive or grow. Therefore, HAT medium can be used to screen for successfully fused cells.

[0041] Cell growth was observed within 5 days after cell fusion, and ELISA plates were coated overnight with 1 μg / mL antigen the day before detection. The next day, the supernatant from the 96-well cell culture plates was aspirated and used as the primary antibody for indirect ELISA detection, 100 μL per well, to determine the presence of specific antibodies in the supernatant. Wells with positive results from indirect ELISA were further cultured in HAT medium until the cells in the wells grew to a medium size and reached a number of 10. 4 At this point, the supernatant from the wells is aspirated for ELISA retesting, and hybridoma cells from wells that show positive results are selected for subcloning.

[0042] (2) Subcloning and detection Subcloning of hybridoma cells from repeat positive wells using limiting dilution: ① First, determine the number of wells with 60% monoclonal coverage per plate and select them for subcloning. Each time, select wells with high positive values ​​for limiting dilution to reduce cell density and prevent excessive cell growth that could cause hybridoma cells to lose their monoclonal nature.

[0043] ② Secondly, after each subcloning, the supernatant was taken for indirect ELISA detection until a monoclonal cell line that can stably secrete positive antibodies was finally screened out.

[0044] (3) Freezing The cell lines that stably secrete positive antibodies, selected during the subcloning stage, were cultured in 10 cm culture dishes to a scale of 2 × 10⁻⁶ cells / year. 6 When the concentration of cells / mL is reached, it can be frozen.

[0045] 5. Preparation of ascites (1) Sensitization of mice: One week before inoculation with hybridoma cell lines, mice were injected intraperitoneally with liquid paraffin, 0.5 mL per mouse.

[0046] (2) Inoculation with hybridoma cells: Culture hybridoma cells to achieve a cell density of 1×10⁻⁶. 6 -2×10 6 Centrifuge at 1000 rpm at a concentration of / ml, discard the supernatant and collect the cells. Resuspend the cell pellet in sterile PBS and inject 0.5 mL into each sensitized mouse via intraperitoneal injection.

[0047] (3) Collecting ascites fluid: Within 15 days after inoculation with hybridoma cells, observe whether the mouse abdomen is distended. Select mice with obvious abdominal distension, disinfect with povidone-iodine and 75% alcohol, and then use a syringe to puncture the mouse abdomen to aspirate all the ascites fluid in the abdominal cavity for subsequent antibody purification.

[0048] 6. Antibody purification The collected ascites fluid was pretreated before antibody purification. The ascites fluid was centrifuged at 3000 rpm for 10 min to remove adipose tissue and other impurities. The monoclonal antibody was then precipitated using the saturated ammonium sulfate precipitation method and dialyzed overnight in 1×PBS in a dialysis bag. The fluid was then filtered through a 0.45 μm filter membrane the next day for use.

[0049] Monoclonal antibody purification was performed using Protein G agarose affinity chromatography. The specific steps are as follows:

[0050] (1) Affinity chromatography column packing: Take 1 mL of recombinant protein G agarose gel to pre-pack the column, and after the gel precipitates naturally, add 7 column volumes of ultrapure water to wash away the gel preservation solution; (2) Column equilibration: After the ultrapure water has dried naturally, add 7 column volumes of 20 mM / L phosphate buffer (pH 7.4). (3) Sample loading: Add the ascites fluid filtered through a 0.45 μm filter membrane to the purification column; (4) Elution of contaminating proteins: Add 7 column volumes of 20 mM / L phosphate buffer (pH 7.4) to elute contaminating proteins; (5) Antibody elution: The antibody was eluted with 0.1 M / L Tris-HCl (pH 2.7) buffer and the antibody pH was adjusted to neutral with 1 M / L Tris-HCl. The antibody concentration was determined by Nanodrop 2000 and aliquoted and stored at -80℃. (6) Column preservation and regeneration: Elute the gel column with 3-5 column volumes of elution buffer and store at 4°C.

[0051] 7. Monoclonal antibody subtype identification ELISA plates were coated with 1 μg / mL antigen overnight at 4°C. The supernatant of a cell line that could stably secrete positive antibodies was used as the primary antibody, and the secondary antibody was HRP-labeled goat anti-mouse subtypes (IgG1, IgG2a, IgG2b, IgG3, IgM, IgA). After adding the substrate and developing color, the results were compared with the standard results. The comparison results should show that the subtype of the monoclonal antibody corresponds to the subtype of the goat anti-mouse secondary antibody.

[0052] 8. Monoclonal antibody titer detection Monoclonal antibodies purified by Protein G agarose affinity chromatography were subjected to titer testing. The results were determined by calculating the P / N ratio (OD value of positive wells / OD value of negative wells) based on the OD value at 450 nm in the wells, with a P / N ratio ≥ 2.1 as the cutoff value.

[0053] 9. SDS-PAGE Validation and Analysis of Monoclonal Antibody Purification Stacking and separating gels with concentrations of 5% and 12% were prepared. The purified monoclonal antibody was mixed with 4× protein loading buffer and incubated in a 100℃ metal bath for 10 min, followed by electrophoresis at 80 V for 30 min. The voltage was then adjusted to 120 V for the separating gel, and electrophoresis continued for 1.5 h. Coomassie Brilliant Blue R-250 staining was performed for 10 min, and the gel was destained until it became transparent, at which point the results could be observed.

[0054] II. Preparation of Polyclonal Antibodies (1) Preparation of immunogen: Same as in Example 1.

[0055] (2) Animal selection: Two New Zealand white rabbits weighing 1.5-2 kg were used as the immunization group and the control group.

[0056] (3) Immunization procedure: Same as in Example 1.

[0057] (4) Polyclonal antibody purification: Blood was collected from the heart 12 days after the last booster immunization. The serum was allowed to stand naturally and then extracted using the saturated ammonium sulfate precipitation method. The obtained polyclonal antibody was dialyzed overnight in 1×PBS in a dialysis bag. The next day, it was filtered through a 0.45 μm filter membrane and purified using Protein G agarose affinity chromatography column purification method, which was the same as the antibody purification in Example 1.

[0058] III. Establishment of a colloidal gold immunochromatographic method 1. Optimization of reaction conditions (1) Optimal labeling pH value Add 300 μL of colloidal gold solution to a clean, removable 96-well microplate. Adjust the pH using 0.1 M / L K₂CO₃ as shown in Table 1. Then add 9 μL of 1 mg / mL monoclonal antibody, mix well, and incubate at 37°C for 1 h. Finally, add 30 μL of 10% NaCl (w / v) solution, incubate at 37°C for 10 min, and then incubate at room temperature for 2 h. The lowest pH at which the monoclonal antibody maintains stable color without change when binding to the colloidal gold particles is considered the optimal binding pH.

[0059] Table 1. Determination of the optimal pH for monoclonal antibody-labeled colloidal gold. (2) Optimal labeling concentration 300 μL of 10 nM colloidal gold solution was added to a clean, removable 96-well microplate. The pH was adjusted to the optimal level using 0.1 M / L K₂CO₃. Then, different volumes of 1 mg / mL monoclonal antibody were added as shown in Table 2, resulting in monoclonal antibody concentrations of 0 μg / mL, 2 μg / mL, 4 μg / mL, 6 μg / mL, 8 μg / mL, 10 μg / mL, 15 μg / mL, 20 μg / mL, and 30 μg / mL. The mixture was thoroughly incubated at 37°C for 1 h. Then, 30 μL of 10% NaCl solution (w / v) was added, and the plate was incubated at 37°C for 10 min, followed by incubation at room temperature for 2 h. The lowest monoclonal antibody concentration that maintained stable color when bound to the colloidal gold particles was considered the optimal labeling concentration.

[0060] Table 2 Determination of Optimal Labeling Concentration for Clonal Antibodies 2. Large-scale preparation of colloidal gold-labeled monoclonal antibodies Large-scale preparation of gold-labeled monoclonal antibodies was carried out according to the selected optimal pH and labeling concentration.

[0061] 3. Test strip pretreatment The pretreatment methods for the sample pad, gold label pad, and NC membrane were performed in accordance with Qian Hong. Establishment and Application of Colloidal Gold Diagnostic Methods for Brucellosis and Lyme Disease [D]. Shihezi: Shihezi University, 2015.

[0062] 4. Selection of optimal coating concentration for the detection line The T line was prepared by coating an NC membrane with rabbit anti-Listeria monocytogenes polyclonal antibody at a known concentration. Different coating concentrations for the T line were set as follows: 0.5 mg / mL, 1.0 mg / mL, 1.5 mg / mL, and 2.0 mg / mL. The C line was coated with goat anti-mouse IgG (specifically for the C line) at a concentration of 2.0 mg / mL. Both the T line and the C line were labeled using a single-dimensional planar spot gold sputtering instrument at an antibody labeling volume of 1 μL / cm. The colloidal gold test strips were assembled and tested. The optimal antibody coating concentration for the test line was determined based on the results.

[0063] 5. Assembly of colloidal gold test strips The gold-labeled pad was placed in the prepared colloidal gold-labeled monoclonal antibody solution, soaked for 30 min, then removed and air-dried before being cut into 8 mm wide pieces for later use. Detection lines and control lines were marked on the NC membrane using a single-dimensional planar gold sputtering instrument at a concentration of 1 μL / cm. The absorbent pad was cut into 18 mm wide pieces. Figure 1 As shown, attach each component of the test strip to the PVC base plate in sequence, and then cut it into 4 mm wide test strips using a cutting machine.

[0064] 6. Specificity Listeria monocytogenes and other non-target strains (Staphylococcus aureus, Streptococcus agalactiae, Escherichia coli, Enterococcus haematobium, Streptococcus dysgalactiae, Salmonella, Staphylococcus chromogenic, Staphylococcus stoichioides, Streptococcus lactis) were cultured, and the activated bacterial solution was loaded onto the assembled test strip. The results were observed after standing on the table for 10 minutes to evaluate the specificity of the test strip.

[0065] 7. Sensitivity Listeria monocytogenes was cultured and its total colony count was calculated. After 10-fold serial dilution, the bacterial solutions of each dilution were loaded onto the prepared colloidal gold test strips. The results were observed after standing on the table for 10 minutes to evaluate the sensitivity of the test strips.

[0066] result I. Preparation of Monoclonal Antibodies against Listeria monocytogenes 1. Detection of antibody titer in mouse serum Using Listeria monocytogenes as an immunogen, five mice were immunized four times. The mouse serum was then analyzed by indirect ELISA, and the results are shown in Table 3. All four mice exhibited a strong immune response. Even at a serum dilution of 1:128000, the P / N ratio (OD value of positive serum at 450 nm / OD value of negative serum at 450 nm) remained high, all greater than 2.1, indicating that the mice had met the requirements for cell fusion after four immunizations. In particular, mouse number 1 had a P / N ratio of 22.045 at a serum dilution of 1:128000, significantly higher than the other mice. Therefore, mouse number 1 was selected for subsequent cell fusion.

[0067] Table 3. Mouse serum titers 2. Fusion Screening After cell fusion, the supernatant of the fused cells was analyzed using indirect ELISA to screen for positive clones. Based on the indirect ELISA results, 18 positive clones were initially screened. These 18 positive clones were retested three times, and finally, four subclones with a 100% positive rate were identified. These four cell lines were then further cultured and cryopreserved. The 1H6 1B8 cell line showed a significant advantage in titer compared to the other three cell lines, and therefore, the 1H6 1B8 cell line was selected for subsequent large-scale preparation of monoclonal antibodies.

[0068] Table 4. Titer of Supernatant from Positive Clonal Cell Lines 3. Monoclonal antibody subtype identification The mouse monoclonal antibody subtype identification ELISA kit instructions were followed to identify the subtypes. The results are shown in Table 5. The cell lines 5H5 3G7 and 5H6 4D1 selected in this study are IgG1 subtype, while 7C12 2F11 and 1H6 1B8 are IgG2b subtype.

[0069] Table 5. Results of Monoclonal Antibody Subtype Identification 4. Monoclonal antibody titer The purified antibody titer was detected using an indirect ELISA method, and the results are as follows: Figure 2 As shown, the dilution of the positive well with an OD value greater than 2.1 times the set negative control OD value was defined as the titer of the purified monoclonal antibody. The results showed that the negative control OD value at 450 nm was 0.075. When the monoclonal antibody dilution was 1:384000, the OD value of the positive well was 0.1397, with a P / N ratio < 2.1. When the monoclonal antibody dilution was 1:192000, the OD value of the positive well was 0.2812, with a P / N ratio > 2.1. Therefore, the titer of the monoclonal antibody was determined to be 1:192000.

[0070] 5. SDS-PAGE validation and analysis of purified monoclonal antibodies After the purified monoclonal antibody was mixed with 4× protein loading buffer, the mixture was incubated in a 100℃ metal bath for 10 min, followed by SDS-PAGE gel electrophoresis. The target band and protein marker were excised and stained with Coomassie Brilliant Blue. The results are as follows: Figure 3 As shown, the purified monoclonal antibody showed no contaminating proteins, and the bands were clear and the size was as expected.

[0071] II. Preliminary Establishment of Colloidal Gold Immunochromatography Method 1. Optimal pH value for labeling When the monoclonal antibody labeling amount was 9 μg, the pH was adjusted using 0.1 M / L K2CO3, and the results were as follows. Figure 4 The results showed that the colloidal gold solution remained red at pH 6 and above, therefore the optimal pH for the label was chosen to be 6.0.

[0072] 2. Optimal labeling concentration Under optimal pH conditions, different amounts of 1 mg / mL monoclonal antibody were added to colloidal gold solution, and the results were as follows: Figure 5 As shown, the optimal labeling concentration of the monoclonal antibody is 4 μg / mL, corresponding to an optimal labeling amount of 4 μg.

[0073] 3. Optimal coating concentration for the detection line The results of the screening for optimal coating concentration for the detection line are as follows: Figure 6 As shown, when the control line C is coated with 2.0 mg / mL goat anti-mouse IgG and the test line is coated with 2.0 mg / mL antibody, the color of the test strip T line is better than other concentrations. Therefore, 2.0 mg / mL antibody coating amount is selected as the optimal coating amount for the test line.

[0074] 4. Specificity of colloidal gold immunochromatography The specificity test results of the colloidal gold test strip are as follows: Figure 7 As shown, a distinct red line appears on the colloidal gold test strip only when the target strain Listeria monocytogenes is detected, while other non-target strains do not, indicating that the colloidal gold test strip established in this study has good specificity.

[0075] 5. Sensitivity of colloidal gold immunochromatography The total bacterial count was 1×10⁻⁶. 8 Listeria monocytogenes CFU / mL was serially diluted 10-fold. The diluted bacterial solutions were added to the prepared test strips, allowed to stand for 10 minutes, and then the results were observed. The results are as follows: Figure 8 As shown, when the bacterial concentration is 1×10 6 At CFU / mL, a clearly visible red line appears on the test line, while the bacterial concentration is 1×10⁻⁶. 5 At CFU / mL, a red line appeared on the test line, but it was not very obvious. Therefore, the detection limit of the colloidal gold test strip can be determined to be 1×10⁻⁶. 5 CFU / mL.

[0076] It should be noted that when numerical ranges are mentioned in the claims of this invention, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected. To avoid redundancy, the present invention describes preferred embodiments.

[0077] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

[0078] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A colloidal gold immunochromatographic test strip for Listeria monocytogenes, the test strip comprising a base plate and a sample pad, a gold label pad, a detection pad, and an absorbent pad sequentially disposed on the base plate, characterized in that, The gold label pad is labeled with a monoclonal antibody against Listeria monocytogenes, the monoclonal antibody against Listeria monocytogenes being 1H6 1B8, and the labeling concentration being 4-30 μg / mL. The nucleotide sequence of the heavy chain variable region of 1H6 1B8 is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.

2. The nucleotide sequence of the light chain variable region of 1H6 1B8 is shown in SEQ ID NO.3, and the amino acid sequence is shown in SEQ ID NO.

4.

2. The colloidal gold immunochromatographic test strip according to claim 1, characterized in that, The pH of the gold-labeled pad is 6-9.

5.

3. The colloidal gold immunochromatographic test strip according to claim 1, characterized in that, The amount of antibody coated on the test line of the test pad is 0.5-2.0 mg / mL.

4. A method for preparing the colloidal gold immunochromatographic test strip according to claim 1, characterized in that, Includes the following steps: Under conditions of pH 6-9.5, colloidal gold and Listeria monoclonal antibody were added and incubated. The concentration ratio of colloidal gold to Listeria monoclonal antibody was 10 nM: 2 μg / mL~30 μg / mL. Set the polyclonal antibody against Listeria monocytogenes as the detection line; Assembly of colloidal gold test strips.

5. The preparation method according to claim 4, characterized in that, The method for preparing the monoclonal antibody against Listeria monocytogenes includes the following steps: Mice were immunized with a mixture of inactivated Listeria monocytogenes and Freund's adjuvant. Spleen cells and myeloma cells from immunized mice were mixed at a ratio of 5-10:1 to obtain hybridoma cells through cell fusion. Positive hybridoma cells capable of producing Listeria monoclonal antibodies were inoculated into the peritoneal cavity of mice, and the ascites fluid was collected and purified to obtain the monoclonal antibodies against Listeria monoclonal.

6. The preparation method according to claim 4, characterized in that, The assembly of the colloidal gold test strip includes the following steps: Place the gold-labeled pad in colloidal gold-labeled monoclonal antibody and soak it for later use; Mark the test lines and control lines on the nitrocellulose membrane; The sample pad, gold label pad, and nitrocellulose membrane were sequentially placed on a polyethylene chloride substrate.

7. The application of the colloidal gold immunochromatographic test strip according to claim 1 in the detection of Listeria monocytogenes.

8. The application according to claim 7, characterized in that, The test solution is loaded onto the colloidal gold immunochromatographic test strip, and the results are observed and determined after standing for 8 to 12 minutes.

9. The application according to claim 7, characterized in that, The criteria for judgment are: Negative: If only the control line shows a visible band, while the test line shows no visible band, the test result is negative, indicating that the sample does not contain Listeria monocytogenes. Positive: If visible bands appear on both the test line and the control line, the test result is positive, indicating that the sample contains Listeria monocytogenes. Failure: If no visible band appears on the quality control line, the test is considered invalid regardless of whether a band appears on the test line.

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