Hybridoma cell line secreting an ActA monoclonal antibody and use thereof

Abstract

A hybridoma cell line secreting an ActA monoclonal antibody and a use thereof. The hybridoma cell line was deposited on Jul. 8, 2021, at the China Center for Type Culture Collection (CCTCC) with the depository accession number of CCTCC NO: C2021174. The present invention also discloses the ActA monoclonal antibody secreted by this hybridoma cell line or its progeny cell line, a detection kit containing this ActA monoclonal antibody, immunomagnetic beads, the preparation method and use of immunomagnetic beads, and competitive ELISA and indirect ELISA detection methods. The ActA monoclonal antibody of the present invention has the advantages of high titer, good specificity, and strong affinity with the natural antigen. The Listeria competitive ELISA detection kit and immunomagnetic beads developed based on this antibody have high sensitivity and good stability, effectively monitoring the level of ActA antibodies in clinical serum samples, and can be used for labeling Listeria monocytogenes.

Claims

1. A competitive ELISA detection kit for detecting Listeria monocytogenes infection, comprising an Actin assembly-inducing protein (ActA) monoclonal antibody or a polynucleotide; wherein the ActA monoclonal antibody comprises a heavy chain and a light chain, wherein the light chain comprises three complementarity determining regions with amino acid sequences shown as SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively; and the heavy chain comprises three complementarity determining regions with amino acid sequences shown as SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively; wherein the polynucleotide encodes the ActA monoclonal antibody, wherein a nucleotide sequence encoding the light chain of the ActA monoclonal antibody is shown as SEQ ID NO: 8, and a nucleotide sequence encoding the heavy chain of the ActA monoclonal antibody is shown as SEQ ID NO: 10; wherein the competitive ELISA detection kit further comprises one or more of the following: an enzyme-labeled antibody, a coated antigen, a positive and/or negative control serum for Listeria monocytogenes, a coating buffer, a blocking buffer, a dilution buffer, a washing buffer, a substrate solution, and a reaction stop solution.

2. The competitive ELISA detection kit of claim 1, wherein the enzyme-labeled antibody is obtained by labeling the ActA monoclonal antibody against Listeria monocytogenes with horseradish peroxidase; wherein the enzyme-labeled antibody is diluted at a ratio of 1:32000, the coated antigen is rHis-ActA protein, a concentration of the coated antigen is 2.0 g/mL, the positive and/or negative control serum for Listeria monocytogenes is diluted at ratios of 1:8, 1:10, 1:12, 1:16, or 1:20, the coating buffer is carbonate buffer with a pH of 9.6, the blocking buffer is a PBS solution containing 2% to 5% skim milk, the dilution buffer is a PBS solution containing 1-2% BSA, the washing buffer is a PBST solution with a pH of 7.2, the substrate solution is TMB single-component substrate solution, and the reaction stop solution is 0.5-2 mol/L H.sub.2SO.sub.4.

3. The competitive ELISA detection kit of claim 2, wherein the competitive ELISA detection kit comprises 800 L of positive control serum for Listeria monocytogenes, 800 L of negative control serum for Listeria monocytogenes, 450 L of enzyme-labeled antibody diluted 1:640, 30 mL of antibody dilution buffer (1), 60 mL of PBST washing buffer (20), 22 mL of TMB single-component substrate solution, and 12 mL of stop solution.

4. The competitive ELISA detection kit of claim 1, wherein the coating antigen is coated at 4 C. for 16 hours, a blocking time is 3 hours, an incubation time for the enzyme-labeled antibody is 1 hour, and a color development time is 3 minutes.

5. The competitive ELISA detection kit of claim 1, wherein a critical value for the competitive ELISA kit is 27.5%.

Description

BRIEF DESCRIPTION OF DRAWINGS

(1) FIG. 1 shows laser confocal microscopy results of ActA monoclonal antibody.

(2) FIG. 2 shows an effect of different serum dilutions on inhibition rate and P/N value in competitive ELISA.

(3) FIG. 3 shows an effect of different coating temperatures and durations of time on inhibition rate and P/N value.

(4) FIG. 4 shows an effect of different blocking buffers on inhibition rate and P/N value.

(5) FIG. 5 shows an effect of different durations of blocking time on inhibition rate and P/N value.

(6) FIG. 6 shows an effect of different durations of incubation time of enzyme-labeled antibody on inhibition rate and P/N value.

(7) FIG. 7 shows an effect of different durations of color development time on inhibition rate and P/N value.

(8) FIG. 8 shows specificity and sensitivity intersection of the competitive ELISA assay.

(9) FIG. 9 shows an ROC analysis curve of the competitive ELISA assay.

(10) FIG. 10 shows specificity of the competitive ELISA assay.

(11) FIG. 11 shows an effect of different durations of storage time of the solid-phase carrier at 37 C. on detection results.

(12) FIG. 12 shows an effect of different durations of storage time of enzyme-labeled antibody at 37 C. on detection results.

(13) FIG. 13 shows an effect of different durations of storage time of control sera at 37 C. on detection results.

(14) FIG. 14 shows an effect of different durations of coupling time of the antibody on capture efficiency.

(15) FIG. 15 shows an effect of different antibody amounts on capture efficiency.

(16) FIG. 16 shows an effect of different durations of enrichment time of bacterial solution on capture efficiency.

DETAILED DESCRIPTION OF THE INVENTION

(17) The technical solution of the present invention is further described below in conjunction with the accompanying drawings. Unless otherwise indicated, the reagents, methods, and apparatus employed in the present invention are conventional reagents, methods, and apparatus in the art.

(18) 1. Strains and Antibodies:

(19) Listeria monocytogenes, Salmonella typhimurium, Staphylococcus aureus, Escherichia coli, and Vibrio parahaemolyticus are all preserved in our laboratory. The monoclonal antibody against Listeria monocytogenes is prepared by our laboratory.

(20) 2. Main Reagents and Instruments

(21) Carboxylated magnetic beads (PM3-020) were purchased from Shanghai Allrun Nano Science & Technology Co., Ltd; carbodiimide (EDC) was purchased from Shanghai Yuanye Bio-Technology Co., Ltd.; N-hydroxysuccinimide (NHS) was purchased from Shanghai Jiachen Chemical Co., Ltd.; UVM modified Listeria enrichment medium, Fraser base medium, brain heart infusion (BHI) medium, and buffered peptone water were purchased from BD Company; Listeria chromogenic medium was purchased from CHROMagar Company in France; multifunctional magnetic separator was purchased from Shanghai Allrun Nano Material Technology Co., Ltd.

Example 1: Acquisition of ActA Monoclonal Antibody 3G11

(22) 1. Animal Immunization

(23) The specific immunization steps are as follows: 8-week-old female BALB/c mice were immunized with purified His-tagged ActA protein, namely rHis-ActA protein. For the first immunization, rHis-ActA protein was mixed with Freund's complete adjuvant at a volume ratio of 1:1, thoroughly emulsified, and administered via intraperitoneal injection at a dose of 50 g per mouse. Fourteen days later, the second immunization was conducted using Freund's incomplete adjuvant instead of Freund's complete adjuvant, with the remaining steps identical to the first immunization. Seven days after the second immunization, mouse serum was collected, and the antibody titer of ActA in the serum was detected by indirect ELISA. The mice with the highest antibody level were selected for a booster immunization. Fourteen days after the second immunization, the booster immunization was conducted by intraperitoneally injecting rHis-ActA protein into the mice with the highest antibody level at a dose of 100 g per mouse. Three days after the booster immunization, spleen lymphocytes were collected from the mice for cell fusion.

(24) The rHis-ActA protein was sourced from freshly cultured recombinant E. coli BL21(DE3) (pET-actA), which was inoculated at a 1:50 ratio into LB liquid medium containing kanamycin, and shaken at 37 C. until the OD value reached 0.4; then 0.1 mM/L protein inducer (IPTG) was added, and the culture was incubated at 37 C. for 6 hours. The bacterial cells were collected by centrifugation, washed with sterile PBS, and resuspended. The bacterial cells were lysed in an ice bath using an ultrasonic cell disruptor (800 W, 10 seconds for each ultrasonication, 20 seconds for each interval). The lysate was centrifuged, and the supernatant was collected and added to an affinity chromatography column (His-Bind purification kit, purchased from Novagen) pre-washed with 20 mL of sterile pure water. Binding buffer (20 mL), washing buffer (12 mL), and elution buffer (12 mL) were sequentially added, and the purified product rHis-ActA was collected in a sterile finger-shaped tube. The binding buffer, washing buffer, and elution buffer were all included in the His-Bind purification kit.

(25) 2. Cell Fusion

(26) The specific steps are as follows: on the third day after the booster immunization in Example 1, the immunized mice were euthanized following biosafety procedures. The mice were soaked in 75% ethanol for 5 minutes for disinfection, and spleen cells were aseptically collected and fused with log-phase myeloma cells SP2/0 by using PEG (MW1450). Peritoneal macrophages from ICR mice (purchased from the Comparative Medicine Center of Yangzhou University) were used as feeder cells. The fused cells and feeder cells were suspended in HAT medium, dispensed into a 96-well plate, and cultured in a 37 C., 5% CO2 incubator. Fresh HAT medium was added after 5 days, and HT medium was used after 10 days. On a regular basis, the cultures were observed, media were changed, and assays were performed.

(27) 3. Establishment of Indirect ELISA Detection Assay

(28) The indirect ELISA assay was used to screen positive cell clones, and the optimal coating buffer concentration for the detection antigen rGST-ActA was determined by matrix test. The detection antigen rGST-ActA was diluted in a horizontal gradient with coating buffer, and the diluted detection antigen rGST-ActA was added, with 100 L per well, to coat the ELISA plate overnight at 4 C. The plate was washed three times with PBST, and 300 L of PBS containing 2% BSA was added to each well as a blocking buffer, and incubated overnight at 4 C. The immune mouse serum was serially diluted 1:100, 1:200, 1:400, 1:800, 1:1000, and 1:2000 with blocking buffer, which was then added to the plate at 100 L per well. Serum from SPF mice diluted similarly was used as a negative control. The plate was incubated at 37 C. for 2 hours. After washing three times with PBST, HRP-labeled goat anti-mouse IgG antibody diluted 1:5000 with blocking buffer was added to the plate at 100 L per well, and the plate was incubated at 37 C. for 1 hour. After washing, 100 L of TMB substrate was added to each well for color development for 5 minutes. The reaction was stopped by adding 50 L of 2M H.sub.2SO.sub.4, and the absorbance at 450 nm was measured using a microplate reader. The optimal conditions were determined when the negative serum has its maximum OD.sub.450 value and the positive serum has an OD.sub.450 value around 1.0.

(29) The source of rGST-ActA is as follows: freshly cultured recombinant E. coli BL21 (pGEX-6p-1-actA) was inoculated at a ratio of 1:50 into LB liquid medium containing ampicillin, shaken at 37 C. until the OD value reached 0.4, and induced with 0.2 mM/L IPTG at 30 C. for 5 hours. The bacterial cells were collected by centrifugation, washed with sterile PBS, and resuspended. The cells were lysed in an ice bath using an ultrasonic cell disruptor (800 W, 10 seconds for each ultrasonication, 20 seconds for each interval). The lysate was centrifuged, and the supernatant was collected and added to a Sepharose 4B column pre-washed with 10 mL of pre-cooled PBS. The column was equilibrated with pre-cooled PBS, and 2 mL of elution buffer was added. The purified product rGST-ActA was collected in a sterile fingure-shaped tube (refer to the Redipack GST purification moduce steps, purchased from Amersham Bioscience).

(30) 4. Screening Positive Clones

(31) The established indirect ELISA assay was used to detect antibodies secreted by hybridoma cells. The specific method is as follows: the culture supernatant of hybridoma cells was added to a pre-coated ELISA plate, at 100 L per well. SP2/0 cell supernatant was used as a negative control, and immune mouse polyclonal serum as a positive control. The plate was incubated in a 37 C. water bath for 2 hours and washed three times with PBST. HRP-labeled goat anti-mouse IgG antibody diluted 1:5000 with blocking buffer was added to the plate, at 100 L per well, and incubated in a 37 C. water bath for 1 hour. After washing, TMB substrate was added for color development for 5 minutes, and the absorbance at 450 nm was read. Wells with OD.sub.450 readings more than twice the negative control were considered positive. One positive clone was named positive cell clone 3G11. This positive cell clone 3G11 was deposited on Jul. 8, 2021, at the China Center for Type Culture Collection (CCTCC), located at Wuhan University, China, with the depository accession number of CCTCC NO: C2021174, and the classification name of Hybridoma Cell Line 3G11.

(32) 5. Cloning Positive Hybridoma Cells

(33) The positive cell clone 3G11 was diluted by the limiting dilution method, then subcloned three times, and preserved. The positive cell clone 3G11 corresponds to the hybridoma cell line with the depository accession number of C2021174.

Example 2: Preparation of ActA Monoclonal Antibody Against Listeria monocytogenes

(34) 1. Preparation of Ascites

(35) Ten-week-old female BALB/c mice were selected for the preparation of ascites. Liquid paraffin was used as the inducer and injected intraperitoneally into the mice at a dose of 500 L per mouse. One week later, each mouse was injected with 510.sup.5 hybridoma cells. When the mice's abdomens became visibly distended, the ascites fluid was collected. The collected ascites fluid was centrifuged at room temperature to remove precipitates, and the supernatant was collected and stored at 70 C. The monoclonal antibody secreted by the hybridoma cell line with the depository accession number of C2021174 or its progeny cell line (corresponding to hybridoma cell 3G11) was designated as monoclonal antibody 3G11.

(36) 2. Determination of Ascites Titer

(37) The ascites fluid containing 3G11 monoclonal antibody was diluted with blocking buffer at serial ratios of 1:1000, 1:2000, 1:4000, 1:8000, 1:16000, 1:32000, 1:64000, 1:128000, 1:256000, 1:512000, 1:1024000, and 1:2048000, and was added to an ELISA plate coated with detection antigen rGST-ActA at 100 L per well. Ascites fluid containing SP2/0 cells, diluted at the same ratios, was used as a negative control. The plate was incubated at 37 C. for 2 hours and then washed three times with PBST. A working solution of HRP-labeled goat anti-mouse IgG antibody, diluted 1:5000 with blocking buffer, was added to the plate at 100 L per well and incubated at 37 C. for 1 hour. After washing with PBST, TMB substrate was added for color development. After stopping the reaction, the absorbance at 450 nm was read and the results were analyzed. The titer of the monoclonal antibody ascites was determined using a P/N value 2.1 as the criterion. The results showed that the titer of monoclonal antibody 3G11 ascites reached 110.sup.5.

(38) 3. Purification and Labeling of the Monoclonal Antibody

(39) The prepared monoclonal antibody 3G11 ascites fluid was purified using the Protein G affinity chromatography method. The purified 3G11 monoclonal antibody was then labeled with horseradish peroxidase (this step was completed at GenScript, Nanjing).

(40) 4. Sequencing of Listeria monocytogenes Monoclonal Antibody

(41) After identification, the results showed that the amino acid sequence of the complementarity determining region 1 (CDR1) of the light chain of monoclonal antibody 3G11 was shown as SEQ ID NO.1, and specifically was RSSQSLVHSNGNTYLH.

(42) The amino acid sequence of the complementarity determining region 2 (CDR2) of the light chain of monoclonal antibody 3G11 was shown as SEQ ID NO.2, and specifically was RVSNRFS.

(43) The amino acid sequence of the complementarity determining region 3 (CDR3) of the light chain of monoclonal antibody 3G11 was shown as SEQ ID NO.3, and specifically was SQSTHVPYT.

(44) The amino acid sequence of the complementarity determining region 1 (CDR1) of the heavy chain of monoclonal antibody 3G11 was shown as SEQ ID NO.4, and specifically was NYWIG.

(45) The amino acid sequence of the complementarity determining region 2 (CDR2) of the heavy chain of monoclonal antibody 3G11 was shown as SEQ ID NO.5, and specifically was DIYPGTGYTNYNEKFKG.

(46) The amino acid sequence of the complementarity determining region 3 (CDR3) of the heavy chain of monoclonal antibody 3G11 was shown as SEQ ID NO.6, and specifically was DLDY.

(47) The full-length amino acid sequence of the light chain was shown as SEQ ID NO. 7, and specifically was

(48) TABLE-US-00001 MKLPVRLLVLMFWIPASSSDVVMTQTPLSLPVSLGNQASISCRSSQSLVH SNGNTYLHWYLQKPGQSPKLLIYRVSNRFSGVPDRFSGSGSGTDFTLKIS RVEAEDLGVYFCSQSTHVPYTFGGGTKLEIK.

(49) The nucleotide sequence encoding the light chain was shown as SEQ ID NO. 8, and specifically was

(50) TABLE-US-00002 ATGAAGTTGCCTGTTAGGCTGTTGGTGCTGATGTTCTGGATTCCTGCTTC CAGCAGTGATGTTGTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTC TTGGAAATCAAGCCTCCATCTCTTGCAGATCTAGTCAGAGCCTTGTACAC AGTAATGGAAACACCTATTTACATTGGTACCTGCAGAAGCCAGGCCAGTC TCCAAAGCTCCTGATCTACAGAGTTTCCAACCGATTTTCTGGGGTCCCAG ACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGC AGAGTGGAGGCTGAGGATCTGGGAGTTTATTTCTGCTCTCAAAGTACACA TGTTCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAA.

(51) The full-length amino acid sequence of the heavy chain was shown as SEQ ID NO. 9, and specifically was

(52) TABLE-US-00003 MEWSGVFIFLLSVTAGVHSQVQLQQSGADLVRPGTSVKMSCKAAGYTFTN YWIGWVKQRPGHGLEWIGDIYPGTGYTNYNEKFKGKATLTADTSSSTAYM QLSSLTSEDSAIYYCARDLDYWGQGTSVTVSS.

(53) The nucleotide sequence encoding the heavy chain was shown as SEQ ID NO. 10, and specifically was

(54) TABLE-US-00004 ATGGAATGGAGCGGGGTCTTTATCTTTCTCCTGTCAGTAACTGCAGGTGT CCACTCCCAGGTCCAGCTGCAGCAGTCTGGAGCTGACCTGGTAAGGCCTG GGACTTCAGTGAAGATGTCCTGCAAGGCTGCGGGATACACCTTCACTAAC TACTGGATAGGTTGGGTAAAGCAGAGGCCTGGACATGGCCTTGAGTGGAT TGGAGATATTTATCCTGGAACTGGTTATACTAACTACAATGAGAAGTTCA AGGGCAAGGCCACACTGACTGCAGACACATCCTCCAGCACAGCCTACATG CAGCTCAGCAGCCTGACATCTGAGGACTCTGCCATCTATTACTGTGCAAG AGACTTGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA.

Example 3: Specific Binding of ActA Monoclonal Antibody to Listeria monocytogenes Surface Protein ActA

(55) 1. Laser Confocal Observation Based on ActA Monoclonal Antibody Labeling Listeria

(56) (1) The ability of ActA monoclonal antibody to bind to the surface ActA antigen of Listeria monocytogenes was further determined using laser confocal microscopy. The steps were as follows: glass slices were pre-added to a 24-well plate. Caco-2 cells and Listeria monocytogenes solution were mixed at a MOI of 20:1 to obtain a mixture, and 1 mL of the mixture was added to each well. After incubating at 37 C. for 7-8 hours, the plate was washed twice with 10 mM PBS solution (pH 7.2).

(57) (2) 400 L of 4% paraformaldehyde was added to each well and fixed for 20 minutes. After washing twice with PBS, 400 L of 0.5% tritox-100 solution was added to each well and permeabilized at 37 C. in a water bath for 20 minutes. The plate was washed three times with 10 mM PBS solution (pH 7.2).

(58) (3) 400 L of PBS solution containing 2-5% BSA was added to each well and blocked at 37 C. for 2-3 hours.

(59) (4) 400 L of ActA monoclonal antibody solution diluted 1:100 with PBS solution containing 2-5% BSA was added to each well. After incubating at 37 C. for 2-3 hours, the plate was washed five times with 10 mM PBS solution (pH 7.2).

(60) (5) 400 L of Goat Anti-Mouse IgG (Alexa Fluor 488) solution diluted 1:200 with PBS solution containing 2-5% BSA was added to each well. After incubating at 37 C. for 2-3 hours, the plate was washed five times with 10 mM PBS solution (pH 7.2).

(61) (6) 400 L of phalloidin solution diluted 1:1000 with PBS solution containing 2-5% BSA was added to each well. After incubating at 37 C. for 1-2 hours, the plate was washed five times with 10 mM PBS solution (pH 7.2).

(62) (7) 400 L of DAPI solution diluted 1:100,000 with PBS solution containing 2-5% BSA was added to each well. After incubating at 37 C. for 15-30 minutes, the plate was washed five times with 10 mM PBS solution (pH 7.2).

(63) (8) The glass slices from the 24-well plate were gently removed with tweezers and mounted with 50% glycerol. Laser confocal observation was then performed, and the results are shown in FIG. 1.

(64) FIG. 1 shows laser confocal observation results of ActA monoclonal antibody. As shown in FIG. 1, Listeria monocytogenes entered the host cells, escaped host autophagy, and utilized host actin aggregation to form comet tails, facilitating intracellular movement and intercellular spread. This process of aggregating host actin requires the involvement of the Listeria surface antigen ActA. This example used ActA monoclonal antibody and Goat Anti-Mouse Alexa Fluor 488 to visualize ActA. Actin cytoskeleton was stained red by phalloidin, and nuclei were stained blue by DAPI after fixation and permeabilization. The results indicated a strong binding ability of ActA monoclonal antibody to the ActA antigen expressed on the cell wall of intracellular Listeria.

Example 4: Establishment of ActA Monoclonal Antibody Competitive ELISA Assay

(65) 1. Determination of the Working Concentrations of Coated Antigen and Enzyme-Labeled Antibody

(66) The matrix titration method was used. The detection antigen rHis-ActA was diluted with coating buffer (0.05 mol/L carbonate buffer, pH 9.6) to final concentrations of 3.0, 2.0, and 1.0 g/mL. The diluted antigen solution was added with 100 L per well and incubated at 4 C. overnight. The wells were then washed three times with washing buffer (PBST solution, pH 7.2) for 5 minutes each time. Each well was added with 300 L of PBS solution containing 2% BSA for blocking at 37 C. for 3 hours. After blocking, the wells were washed three times with a washing buffer for 5 minutes each time. Serum samples were diluted 1:10 with PBS solution containing 2% BSA. The enzyme-labeled antibody, HRP-ActA monoclonal antibody, was diluted with PBS solution containing 2% BSA at ratios of 1:16000, 1:20000, 1:24000, 1:28000, and 1:32000. The diluted serum samples and enzyme-labeled antibody were added to the ELISA plates simultaneously, at 50 L per well, mixed on a shaker at room temperature for 5 minutes, and then incubated at 37 C. for 2 hours. The wells were washed seven times with washing buffer for 5 minutes each time. 100 L of TMB substrate solution was added to each well and incubated at 37 C. in the dark for 5 minutes. The reaction was stopped by adding 50 L of stop solution (2 mol/L H.sub.2SO.sub.4) to each well. The absorbance at 450 nm (OD.sub.450) was measured using a microplate reader. A blank control (containing only the detection antigen and enzyme-labeled antibody without serum) was also set up. The serum inhibition rate (PI) was calculated using the formula: Inhibition rate=(1-OD.sub.450 of test serum/OD.sub.450 of blank control)100%. The optimal working concentrations of the antigen and enzyme-labeled antibody were determined by selecting the antigen concentration and antibody dilution with the highest P/N ratio (P is the positive control inhibition rate, and N is the negative control inhibition rate), the least antigen and enzyme-labeled antibody usage, and the lowest negative inhibition rate. The best coating antigen concentration was found to be 2.0 g/mL, and the optimal dilution of the enzyme-labeled antibody was found to be 1:32000.

(67) 2. Determination of Serum Dilution

(68) The ELISA plate was coated with the antigen at an optimal concentration of 2.0 g/mL, incubated overnight at 4 C., and then washed with PBST solution (pH 7.2). Each well was added with 300 L of blocking buffer (PBS solution containing 2% BSA) and incubated at 37 C. for 3 hours. After washing, serum samples diluted at ratios of 1:8, 1:10, 1:12, 1:16, and 1:20 were mixed with the HRP-ActA enzyme-labeled antibody (with an optimal concentration of 1:32000) at a ratio of 1:1, 100 L of which mixture is added to each well (two parallel groups was set for each ratio of serum samples). The plate was incubated at 37 C. for 2 hours. After washing, TMB substrate solution was added for color development, incubated at 37 C. in the dark for 3 minutes, and then 2M H.sub.2SO.sub.4 stop solution was added. The absorbance at 450 nm (OD.sub.450 nm) was read. The P/N values were calculated and compared with each other, and the trends in inhibition rates of the negative and positive controls were observed, in order to select the appropriate serum dilution. The results are shown in FIG. 2. FIG. 2 shows the effect of different serum dilutions on inhibition rate and P/N value in competitive ELISA. From FIG. 2, it can be seen that the 1:10 serum dilution provided the highest P/N value, making it the optimal dilution to ensure the sensitivity of the method.

(69) 3. Determination of Coating Conditions

(70) Except for a detection antigen concentration of 2 g/mL, an enzyme-labeled antibody dilution of 1:32000, and a serum dilution of 1:10, the remaining experiment steps and parameters were the same as those in 2 Determination of serum dilution. Three sets of coating conditions were established: Group 1 at 4 C. for 12 hours, Group 2 at 4 C. for 16 hours, and Group 3 at 4 C. for 20 hours, with all other operations unchanged. The PI and P/N values of each group were compared to select the coating conditions with lower time cost and higher P/N value as the optimal coating conditions. The results are shown in FIG. 3. FIG. 3 shows the effect of different coating temperatures and durations of time on inhibition rate and P/N value. From FIG. 3, it can be seen that coating at 4 C. for 16 hours provided the lowest negative PI and lower time cost, thus it was selected as the optimal coating condition.

(71) 4. Determination of Blocking Buffer

(72) Except for a detection antigen concentration of 2 g/mL, an enzyme-labeled antibody dilution of 1:32000, a serum dilution of 1:10, and a coating condition of 4 C. for 16 hours, the remaining experiment steps and parameters were the same as those in 2 Determination of serum dilution. Blocking buffers used were 2% BSA, 5% BSA, 2% skim milk, and 5% skim milk, and all other operations remained unchanged. The PI values and P/N values under each condition were compared, and the blocking buffer with the highest P/N value was selected as the optimal blocking buffer. The results are shown in FIG. 4. FIG. 4 shows the effect of different blocking buffers on the inhibition rate and P/N value. As shown in FIG. 4, using 2% skim milk as the blocking buffer resulted in the highest positive PI, the lowest negative PI, and the largest P/N value. Therefore, 2% skim milk was selected as the optimal blocking buffer.

(73) 5. Determination of Blocking Time

(74) Except for a detection antigen concentration of 2 g/mL, an enzyme-labeled antibody dilution of 1:32,000, a serum dilution of 1:10, a coating condition of 4 C. for 16 h, and a blocking buffer of PBS containing 2% skim milk, the remaining experiment steps and parameters were the same as those in 2 Determination of serum dilution. Different blocking times were set to 2 hours, 2.5 hours, 3 hours, and 3.5 hours, respectively, while all other operations remained unchanged. The PI values and P/N values at each time point were calculated, and the time point with the highest P/N value was selected as the optimal blocking time. The results are shown in FIG. 5. FIG. 5 shows the effect of different durations of blocking time on the inhibition rate and P/N value. As shown in FIG. 5, blocking with 2% skim milk for 3 hours resulted in the highest P/N value. Therefore, 3 hours was selected as the optimal blocking time.

(75) 6. Determination of Incubation Time of Enzyme-Labeled Antibody

(76) Except for the optimal conditions determined from previous experiments (detection antigen concentration of 2 g/mL, enzyme-labeled antibody dilution of 1:32000, serum dilution of 1:10, coating condition of 4 C. for 16 hours, blocking buffer of PBS containing 2% skim milk, and blocking time of 3 hours), the experiment was carried out following the steps in 2 Determination of serum dilution. Different incubation times for the enzyme-labeled antibody were set to 0.5 hours, 1.0 hours, 1.5 hours, and 2.0 hours, with all other operations unchanged. The PI values and P/N values at each time point were calculated, and the time point with the highest P/N value was selected as the optimal incubation time for the enzyme-labeled antibody. The results are shown in FIG. 6. FIG. 6 shows the effect of different durations of incubation time of the enzyme-labeled antibody on inhibition rate and P/N value. From FIG. 6, it can be seen that there was no significant difference in P/N values when incubated at 37 C. for 1.0, 1.5, and 2.0 h. Therefore, the shorter incubation time of 1.0 h was selected as the optimal incubation time.

(77) 7. Determination of Color Development Time

(78) Except for the optimal conditions determined (antigen concentration of 2 g/mL, enzyme-labeled antibody dilution of 1:32000, serum dilution of 1:10, coating condition of 4 C. for 16 hours, blocking buffer of PBS containing 2% skim milk, enzyme-labeled antibody incubation time of 1 hour), the experiment was carried out following the steps in 2 Determination of serum dilution. Different color development times were set to 5 minutes, 7 minutes, and 9 minutes, with all other operations unchanged. The PI values and P/N values at each time point were calculated, and the time point with the highest P/N value was selected as the optimal substrate reaction time. The results are shown in FIG. 7. FIG. 7 shows the effect of different durations of color development time on inhibition rate and P/N value. From FIG. 7, it can be seen that when incubated at 37 C. for 3 minutes, the P/N value was highest and the OD.sub.450 nm value of the blank control was close to 1.0. Therefore, 3 minutes was selected as the optimal color development time.

Example 5: Determination of Threshold for ActA Monoclonal Antibody Competitive ELISA Detection Kit

(79) The ActA monoclonal antibody competitive ELISA detection kit included: one antigen-coated plate, 800 L of positive control serum diluted 1:10, 800 L of negative control serum diluted 1:10, 450 L of enzyme-labeled antibody diluted 1:640, 30 mL of antibody dilution buffer (1), 60 mL of PBST washing buffer (20), 22 mL of substrate solution (TMB single-component substrate solution), 12 mL of stop solution (0.5-2 mol/L H.sub.2SO.sub.4), and one instruction manual.

(80) Except for previously determined competitive ELISA reaction conditions (detection antigen concentration of 2 g/mL, enzyme-labeled antibody dilution of 1:32000, serum dilution of 1:10, coating condition of 4 C. for 16 hours, blocking buffer of PBS containing 2% skim milk, and color development time of 3 minutes), the experiment in Example 5 was carried out following the operations in step 2 of Example 4. PI values for each of the 23 positive sheep sera and 35 negative sheep sera (sourced from a sheep farm in Jiangsu Province) were calculated. Data analysis was performed using SPSS Statistics 17.0 software, and a sensitivity and specificity intersection graph was plotted, as shown in FIG. 8. A receiver operating characteristic (ROC) curve was also plotted, using the largest Youden's index (Youden's index=sensitivity+specificity1) to determine the Cut off value for the competitive ELISA, as shown in FIG. 9. FIG. 8 shows a sensitivity and specificity intersection graph for the competitive ELISA assay. As shown in FIG. 8, the trend of sensitivity and specificity varied with the inhibition rate. Specificity initially increased and then decreased with the inhibition rate, reaching a maximum at an inhibition rate of 0.275 (27.5%). Sensitivity consistently remained at 100%, and the intersection point of the two curves indicated the threshold value of the method, i.e., 0.275 (27.5%). FIG. 9 shows a ROC analysis curve of the competitive ELISA assay. The ROC curve and its associated metrics, including the area under the curve (represented by Area), P value (represented by P value.sup.1/4), the threshold (represented by Cut off value), sensitivity, and specificity, are shown. The diagonal line represents the reference line. The area under the ROC curve was tested, and the result showed an AUC of 1.000 with P<0.0001, indicating a statistically significant difference. Combining the Youden's index, as shown in Table 1, the inhibition rate corresponding to the maximum Youden's index was selected as the threshold for this method. When Youden's index was at its maximum, the optimal critical value for the competitive ELISA was 27.5%, with both sensitivity and specificity at 100%. This indicates that when the inhibition rate of a sample is greater than 27.5%, it is determined to be positive; otherwise, it is negative.

(81) TABLE-US-00005 TABLE 1 Youden's index of selected sheep serum samples Cut-off DSn 1-DSp Youden Index 13.5% 0.714 0 0.714 14.5% 0.771 0 0.771 15.5% 0.8 0 0.8 16.5% 0.886 0 0.886 18.5% 0.914 0 0.914 20.5% 0.943 0 0.943 27.5% 1 0 1 34.0% 1 0.043 0.957 37.0% 1 0.087 0.913 40.0% 1 0.13 0.87 42.0% 1 0.174 0.826 45.0% 1 0.217 0.783

Example 6: Specificity Test of ActA Monoclonal Antibody Competitive ELISA Detection Kit

(82) Using the competitive ELISA assay established in Example 5, positive sera from seven common pathogens, including Escherichia coli (E. coli), Salmonella Enteritidis (S. Enteritidis), Salmonella Pullorum (S. Pullorum), Salmonella Gallinarum (S. Gallinarum), Campylobacter jejuni (C. jejuni), Mycobacterium tuberculosis (M. tuberculosis), and Listeria ivanovii (L. ivanovii), were tested. Positive serum (Positive control) and negative serum (Negative control) controls were also set up. The PI values of all serum samples were calculated and compared to the Cut off value of the method in Example 5 to determine the presence of cross-reactivity. The results are shown in FIG. 10. FIG. 10 shows specificity of the competitive ELISA assay. As shown in FIG. 10, the competitive ELISA detection kit demonstrated good specificity with no cross-reactivity with positive sera from other common pathogens.

Example 7: Repeatability Test of ActA Monoclonal Antibody Competitive ELISA Detection Kit

(83) 1. Intra-Batch Repeatability Test

(84) For the intra-batch repeatability experiment, the competitive ELISA method established in Example 5 was used. The same batch of antigen-coated ELISA plates was used, and seven positive sheep serum samples and one negative sheep serum sample (sourced from a sheep farm in Jiangsu Province) were tested over three consecutive days. The experimental data were collected, and the coefficient of variation (CV) for each sample across the three experiments was analyzed. The results are shown in Table 2. The intra-batch coefficient of variation of the competitive ELISA method ranged from 2.55% to 6.80%.

(85) TABLE-US-00006 TABLE 2 Intra-batch repeatability test results for the competitive ELISA Group 1 Group 2 Group 3 (M) (M) (M) M SD CV P1 0.357 0.397 0.411 0.388 0.023 5.85% P2 0.442 0.48 0.519 0.48 0.031 6.54% P3 0.521 0.545 0.583 0.55 0.026 4.64% P4 0.452 0.472 0.491 0.472 0.016 3.35% P5 0.371 0.427 0.433 0.41 0.028 6.80% P6 0.064 0.07 0.074 0.069 0.004 5.93% P7 0.07 0.079 0.075 0.075 0.004 4.93% N1 1.096 1.142 1.211 1.15 0.047 4.11% Blank 1.173 1.167 1.108 1.149 0.029 2.55%
Note: P: Positive Sample; N: Negative Sample
2. Inter-Batch Repeatability Test

(86) For the inter-batch repeatability experiment, the competitive ELISA method established in Example 5 was used. Three batches of the antigen rHis-ActA were prepared. ELISA plates coated with different batches of the antigen were used to test 10 positive sheep serum samples and 2 negative sheep serum samples. The experimental data were collected and the coefficient of variation (CV) was analyzed. The results are shown in Table 3. The inter-batch coefficient of variation of the competitive ELISA method ranged from 1.22% to 14.44%, which is below 15%. These results indicate that the competitive ELISA detection kit has a low degree of variation and good inter-batch repeatability.

(87) TABLE-US-00007 TABLE 3 Inter-batch repeatability test results for the competitive ELISA Group 1 Group 2 Group 3 Sample (M) (M) (M) M SD CV P1 0.189 0.186 0.185 0.186 0.002 1.22% P2 0.293 0.253 0.268 0.271 0.02 7.49% P3 0.321 0.265 0.246 0.277 0.039 14.18% P4 0.296 0.234 0.247 0.259 0.033 12.54% P5 0.386 0.29 0.327 0.334 0.048 14.44% P6 0.341 0.269 0.277 0.296 0.039 13.34% P7 0.08 0.073 0.077 0.077 0.004 4.57% P8 0.08 0.071 0.076 0.075 0.005 5.99% P9 0.083 0.075 0.083 0.08 0.004 5.52% P10 0.072 0.066 0.071 0.07 0.003 4.25% N1 0.775 0.609 0.658 0.681 0.085 12.50% N2 0.777 0.614 0.683 0.691 0.082 11.86% Blank 0.848 0.822 0.797 0.822 0.025 3.08%
Note: P: Positive Samples; N: Negative Samples
3. Repeatability Test by Different Operators

(88) Three different operators, Operator A, Operator B, and Operator C, used the competitive ELISA assay established in Example 5 to test 14 positive sheep serum samples and 10 negative sheep serum samples. The experimental data were collected and the CV was analyzed. The results are shown in Table 4. The coefficient of variation between different operators for the competitive ELISA method ranged from 1.0% to 9.7%. These results indicate that the competitive ELISA detection kit has a low degree of variation and good repeatability across different operators.

(89) TABLE-US-00008 TABLE 4 Repeatability test results for the competitive ELISA by different operators Sample Operator A Operator B Operator C M SD CV P1 0.177 0.2 0.215 0.197 0.016 7.9% P2 0.225 0.247 0.261 0.244 0.015 6.1% P3 0.356 0.356 0.414 0.375 0.027 7.3% P4 0.249 0.292 0.294 0.278 0.021 7.5% P5 0.257 0.282 0.316 0.285 0.024 8.5% P6 0.274 0.294 0.264 0.277 0.012 4.5% P7 0.321 0.382 0.388 0.364 0.030 8.3% P8 0.237 0.269 0.301 0.269 0.026 9.7% P9 0.172 0.203 0.206 0.194 0.015 7.9% P10 0.214 0.208 0.218 0.213 0.004 1.9% P11 0.074 0.077 0.086 0.079 0.005 6.5% P12 0.062 0.062 0.069 0.064 0.003 5.1% P13 0.073 0.077 0.081 0.077 0.003 4.2% P14 0.075 0.08 0.081 0.079 0.003 3.3% N1 0.743 0.736 0.767 0.749 0.013 1.8% N2 0.666 0.646 0.654 0.655 0.008 1.3% N3 0.724 0.701 0.77 0.732 0.029 3.9% N4 0.776 0.73 0.725 0.744 0.023 3.1% N5 0.821 0.774 0.81 0.802 0.020 2.5% N6 0.763 0.706 0.789 0.753 0.035 4.6% N7 0.747 0.73 0.742 0.740 0.007 1.0% N8 0.737 0.712 0.729 0.726 0.010 1.4% N9 0.726 0.717 0.802 0.748 0.038 5.1% N10 0.619 0.616 0.587 0.607 0.014 2.4% Blank 0.934 0.873 1.05 0.952 0.073 7.7%
Note: P: Positive Sample; N: Negative Sample

Example 8: Thermal Stability Test of ActA Monoclonal Antibody Competitive ELISA Detection Kit

(90) 1. Thermal Stability Test Results of Antigen-Coated Solid-Phase Carrier

(91) Using the competitive ELISA method established in Example 5, ELISA plates coated with the detection antigen were placed at 37 C. and tested on days 1, 2, 3, 5, 7, 10, 15, 20, and 25. The results are shown in FIG. 11. FIG. 11 shows the effect of different durations of storage time of the solid-phase carrier at 37 C. on detection results. As shown in FIG. 11, the CV for the positive, negative, and blank controls was less than 10%, indicating that the antigen-coated solid-phase carrier had good thermal stability.

(92) 2. Thermal Stability Test Results of Enzyme-Labeled Antibody

(93) Using the competitive ELISA method established in Example 5, the enzyme-labeled antibody was diluted with Candor's HRP-Protector at a dilution ratio of 50:1. The diluted enzyme-labeled antibody was placed at 37 C. for accelerated degradation and tested on days 1, 3, 5, 7, 10, 15, 20, and 25. The results are shown in FIG. 12. FIG. 12 shows the effect of different durations of storage time of enzyme-labeled antibody at 37 C. on detection results. As shown in FIG. 12, the CV for the positive, negative, and blank controls was less than 5%, indicating that the enzyme-labeled antibody had good thermal stability.

(94) 3. Thermal Stability Test Results of Negative Control and Positive Control

(95) Using the competitive ELISA method established in Example 5, negative sheep serum and positive sheep serum were diluted with 1% BSA-PBS solution and Candor's enzyme-labeled antibody stabilizer HRP-Protector at a dilution ratio of 17:1:2. The diluted negative and positive sera were placed at 37 C. for accelerated degradation and tested on days 1, 3, 5, 7, 10, 15, 20, and 25. The results are shown in FIG. 13. FIG. 13 shows the effect of different durations of storage time of control sera at 37 C. on detection results. As shown in FIG. 13, the CV for the negative control, positive control, and blank control was less than 5%, indicating that the negative and positive controls had good thermal stability.

Example 9: Application Test I of ActA Monoclonal Antibody Competitive ELISA Detection Kit

(96) The competitive ELISA detection kit described in the present invention was used to test a total of 928 sheep serum samples collected from a Chinese sheep farm. The results showed that antibodies were positive in 205 sheep serum samples and negative in 723 sheep serum samples, with a positive rate of 22.10% for ActA antibodies. This suggests a high infection rate of Listeria monocytogenes in the sheep farm, indicating that preventive and control measures should be implemented.

Example 10: Application Test II of Listeriosis Competitive ELISA Detection Kit

(97) The competitive ELISA detection kit described in the present invention was used to test a total of 306 human serum samples collected from Jiangsu Province (sourced from a local center for disease control and prevention in Jiangsu). The results showed that all 306 serum samples were negative for ActA antibodies.

Example 11: Conformity Test of ActA Monoclonal Antibody Competitive ELISA Detection Kit

(98) The competitive ELISA detection kit described in the present invention was used to test a total of 47 sheep serum samples, of which 25 were positive and 22 were negative. The test results are shown in Table 5. A total of 45 samples had consistent test results, with an overall conformity rate of 95.7%.

(99) TABLE-US-00009 TABLE 5 Conformity results of the competitive ELISA detection kit Diatheva detection kit Positive Negative Total Negative result for the kit of 23 2 25 the present invention Positive result for the kit of 0 22 22 the present invention Total 23 24 47

Example 12: Preparation of Immunomagnetic Beads

(100) First, 200 L of carboxylated magnetic beads were washed three times with 500 L of 0.01 mol/L, pH 6.0 MES-Tween (MEST) solution, and the supernatant was discarded after using a magnetic separator. The beads were resuspended in the MEST solution to obtain a magnetic bead suspension. The activation reagent, which is a MEST solution containing 5 mg/mL of carbodiimide (EDC) and 5 mg/mL of N-hydroxysuccinimide (NHS), was slowly added to the magnetic bead suspension, and the mixture was shaken at 37 C. for 1 hour to obtain activated carboxyl magnetic beads. Then, the ActA monoclonal antibody 3G11 from Example 2 was mixed with the activated carboxyl magnetic beads, and the total volume was adjusted to 500 L using the MEST coupling buffer. The mixture was shaken on a mixing instrument and incubated at 37 C. for 4 hours to obtain immunomagnetic beads. 1 mL of PBST (pH 7.4, 2% BSA) buffer was added for blocking, and incubation was performed at 37 C. for 2 hours. Finally, the supernatant was discarded after using magnetic separation, and the beads were washed three times with PBST, then resuspended in PBST (pH 7.4, 0.02% NaN.sub.3, 0.5% BSA) storage solution, and stored at 4 C.

Example 13: Optimization of Antibody Coupling Time

(101) Equal amounts of activated carboxyl magnetic beads (200 L each), obtained from Example 12, were taken. 100 g of ActA monoclonal antibody 3G11 was added to each group of activated carboxyl magnetic beads, and the total volume was adjusted to 500 L using MEST coupling buffer. The reactions were carried out at 37 C. for 3 hours, 4 hours, and 5 hours, respectively, with three parallels for each group, that is, nine groups of immunomagnetic beads in total. Each of the nine groups of immunomagnetic beads was added to Listeria monocytogenes bacterial solution at a concentration of 10.sup.3 CFU/mL and a volume of 1 mL, and incubated at 37 C. for 1 hour. The beads were washed three times with a sterile PBS solution, followed by magnetic separation on a magnetic stand. The supernatant was discarded, and the immunomagnetic beads were resuspended in 100 L of sterile PBS solution and then spread on BHI agar plates. The capture efficiency of the immunomagnetic beads was calculated based on the results obtained from plate counting, determining the optimal antibody coupling time. The capture efficiency (CE) was calculated using formula (1):

(102) $\begin{array}{cc}CE\left(\%\right)=\left(1-C_u/C_0\right)100,& \left(1\right)\end{array}$ where C0 is the number of colonies before capture, and Cu is the number of colonies in the supernatant after capture.

(103) The results are shown in FIG. 14. FIG. 14 shows the effect of different durations of coupling time of the antibody on capture efficiency, where Colony count represents the number of captured bacteria, and Capture rate represents the capture efficiency. As shown in FIG. 14, as the coupling time of ActA monoclonal antibody 3G11 increased from 3 hours to 4 hours, the number of captured bacteria and the capture efficiency of the immunomagnetic beads also increased. At 4 hours, the number of captured bacteria and the capture efficiency reached their peak. After the coupling time exceeded 4 hours, both the number of captured bacteria and the capture efficiency gradually decreased. Considering the bioactivity of the antibody and time cost, 4 hours was selected as the optimal antibody coupling time for ActA monoclonal antibody 3G11.

Example 14: Optimization of Antibody Coupling Amount

(104) Equal amounts of activated carboxyl magnetic beads (200 L each) obtained from Example 12 were taken, into which 50 g, 100 g, 150 g, and 200 g of ActA monoclonal antibody 3G11 were added respectively. The total volume of each group was adjusted to 500 L using MEST coupling buffer and incubation was performed at 37 C. for 4 hours. Each group of activated carboxyl magnetic beads was set up with three parallels, that is, 12 groups of immunomagnetic beads in total. Each of the 12 groups of immunomagnetic beads was added to Listeria monocytogenes bacterial solution at a concentration of 10.sup.3 CFU/mL and a volume of 1 mL, and incubated at 37 C. for 1 hour. The beads were washed three times with a sterile PBS solution, followed by magnetic separation on a magnetic stand. The supernatant was removed, and the immunomagnetic beads were resuspended in 100 L of sterile PBS solution and then spread on BHI agar plates. The capture efficiency of the immunomagnetic beads was calculated based on the results obtained from plate counting, determining the optimal antibody coupling amount, using the aforementioned formula (1).

(105) The results are shown in FIG. 15. FIG. 15 shows the effect of different antibody amounts on capture efficiency, where Colony count represents the number of captured bacteria, and Capture rate represents the capture efficiency. As shown in FIG. 15, when the amount of ActA monoclonal antibody 3G11 added was between 50-100 g, the number of captured bacteria and the capture efficiency increased with the amount of added antibody. When the amount of ActA monoclonal antibody 3G11 added was between 100-200 g, the number of captured bacteria and the capture efficiency gradually decreased with the amount of added antibody. To ensure the maximum immune response between the antibody and the target antigen without excessive antibody consumption, 100 g was selected as the optimal coupling amount for ActA monoclonal antibody 3G11.

Example 15: Optimization of Bacterial Solution Enrichment Time

(106) The Listeria monocytogenes bacterial solution was diluted to a concentration of 10.sup.3 CFU/mL (with volume of 1 mL). 200 L of immunomagnetic beads obtained from Example 12 were added and incubated at 37 C. for 15 minutes, 30 minutes, 60 minutes, and 90 minutes, respectively. Each group was set up with three parallels, that is, 12 experiment samples in total. The beads were washed three times with a sterile PBS solution, followed by magnetic separation on a magnetic stand. The supernatant was removed, and the immunomagnetic beads were resuspended in 100 L of sterile PBS solution and then spread on BHI agar plates. The capture efficiency of the immunomagnetic beads was calculated based on the results obtained from plate counting, determining the optimal bacterial solution enrichment time, using the aforementioned formula (1).

(107) The results are shown in FIG. 16. FIG. 16 shows the effect of different durations of enrichment time of bacterial solution on capture efficiency, where Colony count represents the number of captured bacteria, and Capture rate represents the capture efficiency. As shown in FIG. 16, the capture efficiency of the immunomagnetic beads for Listeria monocytogenes was relatively highest at an enrichment time of 15 minutes. The capture efficiency decreased slowly and nearly remained unchanged between 15 minutes and 90 minutes. Since the present invention aims to develop a rapid and efficient detection method, 15 minutes was selected as the optimal enrichment time for the immunomagnetic beads and bacterial solution.

Example 16: Specificity Test for Separation of Listeria monocytogenes Using Immunomagnetic Beads

(108) 200 L of immunomagnetic beads obtained from Example 12 were added to a mixture of Listeria monocytogenes solution, Staphylococcus aureus solution, Salmonella typhimurium solution, Escherichia coli solution, and Vibrio parahaemolyticus solution. Each bacterial solution had a concentration of 10.sup.3 CFU/mL and a volume of 0.2 mL, making the total volume of the mixture 1 mL. After shaking incubation at 37 C. for 15 minutes, the supernatant was discarded, and the beads were washed three times with sterile PBS solution. The immunomagnetic beads were resuspended in 100 L of sterile PBS solution and plated on Listeria chromogenic medium. The plate was incubated at 37 C. for 18-24 hours to observe colony growth. The results are shown in Table 6.

(109) TABLE-US-00010 TABLE 6 Specificity of immunomagnetic beads IMBS-Chromogenic/ Bacterial solution Fluorogenic Culture Media Staphylococcus aureus Salmonella typhimurium Escherichia coli Vibrio parahaemolyticus Listeria monocytogenes + Note: +: colony growth on chromogenic medium after immunomagnetic bead separation (Positive reaction); : no colony growth on chromogenic medium after immunomagnetic bead separation (Negative reaction)

(110) As shown in Table 6, only Listeria monocytogenes grew on the Listeria chromogenic medium when the immunomagnetic beads prepared under optimal conditions were added to the mixture of the five different genera of bacteria. Other bacteria did not grow, indicating that the immunomagnetic beads specifically captured Listeria monocytogenes and that this method had high specificity for Listeria monocytogenes.

Example 17: Sensitivity Test for Separation of Listeria monocytogenes Using Immunomagnetic Beads

(111) Listeria monocytogenes solution was diluted to concentrations of 110.sup.5 CFU/mL, 110.sup.4 CFU/mL, 110.sup.3 CFU/mL, 110.sup.2 CFU/mL, and 110.sup.1 CFU/mL. 1 mL of each diluted solution was added in a 1.5 mL centrifuge tube. 200 L of immunomagnetic beads obtained from Example 12 were added to each centrifuge tube. After shaking incubation at 37 C. for 15 minutes, the supernatant was discarded, and the beads were washed three times with sterile PBS solution. The immunomagnetic beads were resuspended in 100 L of sterile PBS solution and plated on Listeria chromogenic medium. The plate was incubated at 37 C. for 18-24 h to observe colony growth. The IMBS-Listeria chromogenic medium was used for the detection of Listeria monocytogenes in the concentration range of 10.sup.1-10.sup.5 CFU/mL. The results are shown in Table 7.

(112) TABLE-US-00011 TABLE 7 Sensitivity of immunomagnetic beads Listeria monocytogenes solution IMBS-Chromogenic/ concentration (CFU/mL) Fluorogenic Culture Media 10.sup.5 + 10.sup.4 + 10.sup.3 + 10.sup.2 +/ 10.sup.1 Note: +: growth of Listeria monocytogenes on Listeria chromogenic medium after immunomagnetic bead separation (Positive reaction); : no growth of Listeria monocytogenes on Listeria chromogenic medium after immunomagnetic bead separation (Negative reaction).

(113) As shown in Table 7, the detection method of the present invention was able to detect Listeria monocytogenes at concentrations of no less than 10.sup.3 CFU/mL. Listeria monocytogenes at concentrations less than 10.sup.2 CFU/mL could not be accurately detected, indicating that the detection limit of this method in the liquid phase is 10.sup.2-10.sup.3 CFU/mL.

Example 18: Capturing Listeria monocytogenes from Actual Samples Using Immunomagnetic Beads

(114) Twenty commercially available chicken meat samples were tested using the established immunomagnetic bead method. Each sample was washed with 25 mL of sterile PBS solution. 12.5 mL of collected PBS solution that washed the samples was used directly for enrichment with immunomagnetic beads obtained from Example 12. After magnetic separation, the beads were washed three times with a sterile PBS solution. The immunomagnetic beads were resuspended in 100 L of sterile PBS solution, plated on a chromogenic medium, and incubated at 37 C. for 18-24 h to observe colony growth. The results were compared with those obtained using traditional microbial culture methods (where the remaining 12.5 mL of PBS solution that washed the chicken meat samples was added to BPW, and Listeria monocytogenes was isolated and identified according to traditional microbial culture methods). The coincidence rate was calculated as follows: (the samples that were positive by both methods+the samples that were negative by both methods)/the total number of samples100%, to evaluate the practical application value of the method. The results are shown in Table 8.

(115) TABLE-US-00012 TABLE 8 Test results of coincidence rate between the immunomagnetic bead and traditional microbial culture method Traditional microbial culture method Positive Negative Total IMBS-Positive 7 0 7 IMBS-Negative 1 12 13 Total 8 12 20

(116) As shown in Table 8, the immunomagnetic bead method detected 7 positive samples and 13 negative samples out of the 20 chicken meat samples tested, with a sample positivity rate of 35.0%. The traditional microbial culture method detected 8 positive samples and 12 negative samples, with a positivity detection rate of 40.0%. The coincidence rate between the immunomagnetic bead method and the traditional microbial culture method was 95.0%, indicating that the immunomagnetic bead method has good concordance and is suitable for clinical detection.

(117) The present invention utilizes immunomagnetic bead (IMBS) technology combined with Listeria chromogenic medium. Specifically, a specific monoclonal antibody 3G11 against the surface protein ActA of Listeria monocytogenes is selected and coupled to carboxylated magnetic beads. Through specific binding with the ActA antigen of Listeria in the sample, the purpose of enriching Listeria is achieved. Compared to conventional Listeria monocytogenes detection techniques, the above-mentioned examples demonstrate specificity, efficiency, and stability in detecting Listeria monocytogenes. The ActA monoclonal antibody 3G11 can specifically couple with carboxylated magnetic beads, directly targeting the antigen without the need for pre-enrichment, reducing false positives and increasing accuracy. The present invention fully combines the advantages of immunomagnetic bead technology and Listeria chromogenic medium, utilizing the high enrichment characteristics of IMBS technology to separate Listeria monocytogenes from the sample, and then using Listeria chromogenic medium for identification. This method has high specificity and can detect Listeria monocytogenes in about 20 h, which saves over 40 hours compared to the traditional national standard method, thus developing a rapid detection method for Listeria monocytogenes. Additionally, the method is simple to operate and does not require large instruments or specially trained personnel, making it suitable for a wide range of applications and potentially serving as technical support for health epidemic prevention and health supervision.

(118) TABLE-US-00013 SequenceListing <110>YangzhouUniversity <120>HybridomacelllinesecretinganActAmonoclonalantibody andusethereof <160>10 <170>SIPOSequenceListing1.0 <210>1 <211>16 <212>PRT <213>ArtificialSequence <400>1 ArgSerSerGlnSerLeuValHisSerAsnGlyAsnThrTyrLeuHis 151015 <210>2 <211>7 <212>PRT <213>ArtificialSequence <400>2 ArgValSerAsnArgPheSer 15 <210>3 <211>9 <212>PRT <213>ArtificialSequence <400>3 SerGlnSerThrHisValProTyrThr 15 <210>4 <211>5 <212>PRT <213>ArtificialSequence <400>4 AsnTyrTrpIleGly 15 <210>5 <211>17 <212>PRT <213>ArtificialSequence <400>5 AspIleTyrProGlyThrGlyTyrThrAsnTyrAsnGluLysPheLys 151015 Gly <210>6 <211>4 <212>PRT <213>ArtificialSequence <400>6 AspLeuAspTyr 1 <210>7 <211>131 <212>PRT <213>ArtificialSequence <400>7 MetLysLeuProValArgLeuLeuValLeuMetPheTrpIleProAla 151015 SerSerSerAspValValMetThrGlnThrProLeuSerLeuProVal 202530 SerLeuGlyAsnGlnAlaSerIleSerCysArgSerSerGlnSerLeu 354045 ValHisSerAsnGlyAsnThrTyrLeuHisTrpTyrLeuGlnLysPro 505560 GlyGlnSerProLysLeuLeuIleTyrArgValSerAsnArgPheSer 65707580 GlyValProAspArgPheSerGlySerGlySerGlyThrAspPheThr 859095 LeuLysIleSerArgValGluAlaGluAspLeuGlyValTyrPheCys 100105110 SerGlnSerThrHisValProTyrThrPheGlyGlyGlyThrLysLeu 115120125 GluIleLys 130 <210>8 <211>393 <212>DNA <213>Artificialsequence <400>8 atgaagttgcctgttaggctgttggtgctgatgttctggattcctgcttccagcagtgat60 gttgtgatgacccaaactccactctccctgcctgtcagtcttggaaatcaagcctccatc120 tcttgcagatctagtcagagccttgtacacagtaatggaaacacctatttacattggtac180 ctgcagaagccaggccagtctecaaagctcctgatctacagagtttccaaccgattttct240 ggggtcccagacaggttcagtggcagtggatcagggacagatttcacactcaagatcagc300 agagtggaggctgaggatctgggagtttatttctgctctcaaagtacacatgttccgtac360 acgttcggaggggggaccaagctggaaataaaa393 <210>9 <211>132 <212>PRT <213>ArtificialSequence <400>9 MetGluTrpSerGlyValPheIlePheLeuLeuSerValThrAlaGly 151015 ValHisSerGlnValGlnLeuGlnGlnSerGlyAlaAspLeuValArg 202530 ProGlyThrSerValLysMetSerCysLysAlaAlaGlyTyrThrPhe 354045 ThrAsnTyrTrpIleGlyTrpValLysGlnArgProGlyHisGlyLeu 505560 GluTrpIleGlyAspIleTyrProGlyThrGlyTyrThrAsnTyrAsn 65707280 GluLysPheLysGlyLysAlaThrLeuThrAlaAspThrSerSerSer 859095 ThrAlaTyrMetGlnLeuSerSerLeuThrSerGluAspSerAlaIle 100105110 TyrTyrCysAlaArgAspLeuAspTyrTrpGlyGlnGlyThrSerVal 115120125 ThrValSerSer 130 <210>10 <211>396 <212>DNA <213>ArtificialSequence <400>10 atggaatggagcggggtctttatctttctcctgtcagtaactgcaggtgtccactcccag60 gtccagctgcagcagtctggagctgacctggtaaggcctgggacttcagtgaagatgtcc120 tgcaaggctgcgggatacaccttcactaactactggataggttgggtaaagcagaggcct180 ggacatggccttgagtggattggagatatttatcctggaactggttatactaactacaat240 gagaagttcaagggcaaggccacactgactgcagacacatcctccagcacagcctacatg300 cagctcagcagcctgacatctgaggactctgccatctattactgtgcaagagacttggac360 tactggggtcaaggaacctcagtcaccgtctcctca396