Double-antibody sandwich ELISA kit for rapidly detecting vibrio parahaemolyticus and application thereof

Abstract

The invention discloses a double-antibody sandwich ELISA kit for rapidly detecting vibrio parahaemolyticus and application of the double-antibody sandwich ELISA kit, an optimal pair 6D11 and 12B1 is obtained through preparation of monoclonal antibodies and screening of different antibody pairs, and a double-antibody sandwich ELISA method for the vibrio parahaemolyticus is established by taking the monoclonal antibody 6D11 as a coating antibody and taking 12B1-HRP as a detection antibody. The method has relatively high sensitivity (1.37 * 10 < 4 > CFU / mL) and relatively good specificity on vibrio parahaemolyticus, and has cross reaction on nine tested vibrio parahaemolyticus strains. And no cross reaction (27 / 29) exists on other 27 common intragenus and extragenus bacteria of vibrio, the detection limit of vibrio parahaemolyticus in seafood after enrichment for 6 hours is 10 CFU / mL, and the detection result is consistent with the detection result of a selective flat plate of a national standard method. The method provides an accurate, reliable and rapid analysis means for detection of vibrio parahaemolyticus in marine products.

Description

Technical Field

[0001] This invention belongs to the field of immunoassay technology, specifically relating to a rapid detection kit for Vibrio parahaemolyticus using a double-antibody sandwich ELISA and its application. Background Technology

[0002] Vibrio parahaemolyticus is a Gram-negative, halophilic zoonotic bacterium widely distributed in marine organisms such as fish, shrimp, and shellfish. It is associated with acute hepatopancreatic necrosis and has caused significant losses to shrimp farming globally. Vibrio parahaemolyticus is also a major causative agent of foodborne illnesses, placing a heavy burden on global public health. Specifically, consuming undercooked seafood or cross-contaminated food can cause acute gastroenteritis, and in severe cases, even sepsis. Thermostable direct hemolysin (TDH) and TDH-associated hemolysin (TRH) are the core virulence factors causing the Kanagawa phenomenon; while heat-labile hemolysin (TLH), although not directly pathogenic, is conserved in most strains. These three genes are used as detection markers for Vibrio parahaemolyticus. In China, from 2010 to 2020, a total of 1,772 outbreaks involving Vibrio parahaemolyticus were reported, with over 27,000 cases. Other high-burden areas, including Japan and Southeast Asia, continue to report localized outbreaks.

[0003] In my country, the limit for Vibrio parahaemolyticus in food is set at 100 MPN / g to 1000 MPN / g in a maximum of one out of five samples, while the levels in other samples must be ≤100 MPN / g. No sample may exceed 1000 MPN / g. Traditional methods for detecting Vibrio parahaemolyticus, including culture-based and nucleic acid-based methods, have limitations in terms of time efficiency, ease of operation, throughput, and field applicability. Immunological detection methods such as enzyme-linked immunosorbent assay (ELISA) and lateral flow immunochromatography (LFIA) offer advantages such as speed, low cost, and minimal equipment dependence, and have been successfully applied to the detection of other foodborne pathogens. However, due to the lack of high-abundance species-specific surface antigens and interference from complex seafood matrices, immunoassays still face challenges in terms of sensitivity and specificity, resulting in a limited number of mature products globally. Existing immunoassay methods targeting heat-stable direct hemolysin-associated hemolysin (TRH), Pir toxin, or LppQ87G48-based methods fall short in comprehensively and unbiasedly detecting all Vibrio parahaemolyticus serotypes or achieving low limits of detection (LOD). Previous reports, primarily based on immunomagnetic bead separation-LFIA systems using commercial polyclonal and monoclonal antibodies (mAbs), have reported low LODs (10 CFU / mL) after the enrichment step; however, these methods have not evaluated the specificity of the antibody or the overall specificity of the method. Therefore, there is an urgent need for a rapid, highly sensitive, and highly specific immunoassay method for Vibrio parahaemolyticus.

[0004] OmpA, the outer membrane protein of Vibrio parahaemolyticus, is its major outer membrane protein and participates in osmotic pressure regulation. High expression of this protein can enhance bacterial resistance to antibiotics by regulating small molecule permeability, thus it is considered a potential candidate molecule for subunit vaccines. Previous comparative genomics and surface proteomics analyses by our research group showed that the three OmpA proteins are conserved at the genus level, while retaining Vibrio parahaemolyticus-specific sequence characteristics, suggesting their potential as reliable immunodiagnostic markers. Notably, OmpA is highly exposed on the bacterial surface under various environmental conditions and is regulated by natural antibacterial agents. Immunological studies have confirmed that recombinant OmpA from Vibrio parahaemolyticus can induce a strong humoral immune response and partial protective immunity in mice, with a reported protection rate as high as 73%. Furthermore, three multivalent vaccine candidates were identified from hybrid OmpA obtained through DNA shuffling technology, which can effectively resist extracellular infection by Vibrio alginolyticus and intracellular infection by Edwardsiella tarda.

[0005] Given OmpA's stable surface exposure, high abundance, strong immunogenicity, and conservation, it is an ideal candidate molecule for developing sensitive and specific immunoassay methods for Vibrio parahaemolyticus. Despite the aforementioned progress, there are still few reports on immunoassay methods using OmpA monoclonal antibodies (mAbs) for the detection of Vibrio parahaemolyticus. Summary of the Invention

[0006] This invention proposes a rapid detection kit for Vibrio parahaemolyticus using a double-antibody sandwich ELISA and its application.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] The first objective of this invention is to provide a hybridoma cell line combination for a monoclonal antibody against Vibrio parahaemolyticus OmpA protein, the hybridoma cell line combination consisting of hybridoma cell line PTD and hybridoma cell line TBZ;

[0009] Among them, hybridoma cell line PTD was deposited at the China General Microbiological Culture Collection Center on November 13, 2025, with accession number CGMCC No. 46748, and classified as a monoclonal cell line; hybridoma cell line TBZ was deposited at the China General Microbiological Culture Collection Center on November 13, 2025, with accession number CGMCC No. 46749, and classified as a monoclonal cell line.

[0010] The second objective of this invention is to provide a monoclonal antibody combination against Vibrio parahaemolyticus OmpA protein, wherein the monoclonal antibody combination consists of monoclonal antibody 6D11 and monoclonal antibody 12B1 secreted by a hybridoma cell line combination.

[0011] The third objective of this invention is to propose the application of monoclonal antibody combinations in the preparation of kits, reagents, or test strips for the detection of Vibrio parahaemolyticus.

[0012] The fourth objective of this invention is to provide a double-antibody sandwich ELISA kit for detecting Vibrio parahaemolyticus, wherein the kit comprises a combination of monoclonal antibodies, wherein monoclonal antibody 6D11 is used as the coating antibody and monoclonal antibody 12B1 is used as the detection antibody.

[0013] Furthermore, the kit also includes standard solutions, blocking solutions, diluents, washing solutions, colorimetric solutions, and stop solutions, as well as PBS sample diluent containing 1% Triton X-100 to achieve high sensitivity and resistance to matrix interference.

[0014] The fifth objective of this invention is to provide a colloidal gold immunochromatographic test strip for detecting Vibrio parahaemolyticus. The test strip is constructed as follows: gold nanoparticles are conjugated with monoclonal antibody 12B1 for binding to the antigen. Monoclonal antibody 6D11 and goat anti-mouse IgG antibody are diluted with 0.01 mol / L phosphate buffer and sprayed onto a nitrocellulose membrane at a spraying rate of 0.9 µL / cm to form a detection line (T line) and a control line (C line), respectively. The NC membrane and sample pad are placed in a 37°C oven for 4 hours. Subsequently, the NC membrane, sample pad, and absorbent pad are cut into 3 mm wide test strips and assembled onto a polyvinyl chloride backing card.

[0015] The sixth objective of this invention is to propose a rapid double-antibody sandwich ELISA method for detecting Vibrio parahaemolyticus, the method of which is as follows:

[0016] S1: Preparation of specific Vibrio parahaemolyticus monoclonal antibody: Using purified soluble recombinant OmpA protein as an immunogen, 6-8 week old BALB / c mice were immunized. After immunization and fusion, cells strongly positive for Vibrio parahaemolyticus were screened for subcloning to obtain Vibrio parahaemolyticus specific monoclonal antibody.

[0017] S2: Screening for paired monoclonal antibodies: Purify the Vibrio parahaemolyticus-specific monoclonal antibodies obtained in step S1, and label them with horseradish peroxidase (HRP). After successful labeling, perform sandwich pairing.

[0018] S3: Establishment of a sandwich ELISA method specific to Vibrio parahaemolyticus: The paired monoclonal antibodies obtained in step S2 are used, with monoclonal antibody 6D11 as the coating antibody and monoclonal antibody 12B1 as the detection antibody. The ELISA plate is coated with monoclonal antibody 6D11 to specifically capture Vibrio parahaemolyticus. The enzyme-labeled antibody 12B1-HRP on the ELISA plate binds to the captured Vibrio parahaemolyticus. After the substrate is added, it is catalyzed by HRP enzyme and produces an absorbance at 450 nm. The result is determined according to the P / N value. Specifically: if Vibrio parahaemolyticus is captured by the coating antibody 6D11 and binds to the enzyme-labeled antibody 12B1-HRP, and catalyzes the substrate to produce an absorbance at 450 nm (P / N ≥ 2.1), it is judged as positive; if the concentration of Vibrio parahaemolyticus is too low (P / N < 2.1), it is judged as negative.

[0019] Furthermore, the specific detection steps in S3 are as follows:

[0020] (1) Coating: Coat the microplate with 4 μg / mL 6D11, using 100 μL / well, and incubate at 37℃ for 2 h;

[0021] (2) Washing: Wash the plate three times with PBST for 3 minutes each time, using 200 μL / well, and then spin dry the reaction plate;

[0022] (3) Sealing: Seal the wells of the plate with a carbonate buffer solution containing 0.2% gelatin, at a rate of 200 μL / well, and seal at 37°C for 2 h;

[0023] (4) Washing: Same as step (2);

[0024] (5) Samples: Vibrio parahaemolyticus bacterial suspension was diluted with PBS containing 1% Triton X-100 at a 3-fold gradient from 10... 7 CFU / mL was serially diluted to 13700 CFU / mL. A blank control containing 1% Triton X-100 in PBS was also set up. 100 μL of sample was added to each well and incubated at 37°C for 1 hour.

[0025] (6) Washing: Same as step (2);

[0026] (7) Add 4 μg / mL enzyme-labeled antibody 12B1-HRP, 100 μL / well, and react at 37℃ for 1 h;

[0027] (8) Washing: Wash the board four times;

[0028] (9) Color development: Add color development solution at a ratio of 1:5 of TMB to substrate solution, with a volume of 100 μL / well, and develop color for 12 minutes;

[0029] (10) Termination: Add 50 μL of termination solution per well;

[0030] (11) Measurement: OD450nm was measured using an enzyme-linked immunosorbent assay (ELISA) reader.

[0031] Furthermore, the pairing parameters included 4 μg / mL of coated antibody 6D11, coating buffer of pH 9.6, 0.01 M carbonate buffer, washing buffer of 0.5% Tween 20 solution prepared with pH 7.2, 0.01 M PBS, and a standard concentration of 10 μg / mL. 7 The concentration of Triton X-100 solution was 1% (CFU / mL), prepared with standard dilution buffer pH 7.2 and 0.01M PBS, and the concentration of enzyme-labeled antibody 12B1-HRP was 4 μg / mL.

[0032] Furthermore, the standards included six Vibrio parahaemolyticus standard strains CICC 21618, CICC 21619, CICC21528, CICC 10552, CGMCC 1.1616, and CICC 21617, as well as three Vibrio parahaemolyticus isolates.

[0033] The above technical solution can achieve the following beneficial effects:

[0034] This invention prepares monoclonal antibodies against Vibrio parahaemolyticus through bacterial immunization. It was found that a double-antibody sandwich ELISA method using monoclonal antibodies 6D11 and 12B1 exhibits good specificity against Vibrio parahaemolyticus. Tests showed that this method had a sensitivity of 1.37 × 10⁻⁶ against nine strains of Vibrio parahaemolyticus. 4 -10 6 The test strip, with a concentration of CFU / mL and no cross-reactivity with 27 other strains, can be used for initial screening of Vibrio parahaemolyticus in food and clinical analyses. The visual detection limit of the constructed test strip is 10. 5 After enrichment for 6 hours, the detection limit can be reduced to 10 CFU / mL.

[0035] This invention has good stability, low cost, and can simultaneously detect large batches of samples, providing an immunological method for detecting Vibrio parahaemolyticus in food and aquatic products.

[0036] This ELISA assay achieved a detection limit of 4.12 × 10⁻⁶ for seven strains of Vibrio parahaemolyticus tested, including six standard strains (CICC 21618, CICC 21619, CICC21528, CICC 10552, CGMCC 1.1616, and CICC 21617) and three food poisoning isolates. 4 CFU / mL, with CICC 21619 showing the lowest detection limit at 1.37 × 10⁻⁶ CFU / mL. 4 CFU / mL. The detection limit of CGMCC1.1616 is relatively high, at 10. 6 CFU / mL. It showed no cross-reactivity with 27 other strains tested, such as Escherichia coli O157, Enterobacter cloacae, Proteus, and Aeromonas. Suitable for large-scale, high-throughput rapid detection of food and clinical samples, it possesses significant economic and social value and is worthy of promotion and application. Attached Figure Description

[0037] Figure 1. Preparation of recombinant OmpA protein from Vibrio parahaemolyticus;

[0038] Figure 2. Results of characterization analysis of monoclonal antibodies;

[0039] Figure 3. Optimization of the double-antibody sandwich ELISA method;

[0040] Figure 4. Detection limit and specificity of the double-antibody sandwich ELISA method;

[0041] Figure 5 shows the detection limit and specificity of the colloidal gold test strip and the detection of actual samples. Detailed Implementation

[0042] The following is in conjunction with the appendix Figure 1-5 The present invention will be further illustrated by the following examples:

[0043] A hybridoma cell line combination for a monoclonal antibody against Vibrio parahaemolyticus OmpA protein, the hybridoma cell line combination consisting of hybridoma cell line PTD and hybridoma cell line TBZ;

[0044] Among them, hybridoma cell line PTD was deposited at the China General Microbiological Culture Collection Center on November 13, 2025, with accession number CGMCC No. 46748, and hybridoma cell line TBZ was deposited at the China General Microbiological Culture Collection Center on November 13, 2025, with accession number CGMCC No. 46749. The deposit address of the above two cell lines is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0045] A monoclonal antibody ensemble against Vibrio parahaemolyticus OmpA protein, the monoclonal antibody ensemble consisting of monoclonal antibody 6D11 and monoclonal antibody 12B1 secreted by a hybridoma cell line ensemble.

[0046] like Figure 1-5 As shown, a rapid double-antibody sandwich ELISA method for detecting Vibrio parahaemolyticus includes the following implementation methods:

[0047] I. Immunogen Preparation

[0048] (1) Strains used: Vibrio parahaemolyticus CICC 21618, recombinant protein: NP_800696.1 OmpA WP005463527.1 (gene) VPA1186) sequence: MNKVAIAVAAVVAGSSALLNSAQAEMYIGGKVGMTTLDDACYLNSPCDDEAFGAGLHIGYDFTDFIGLEYGVDFLGDYKANFKSGASTVDTIDGNLTALTLAPKFNWHLNDSWNLFAKIGGAYMMSEDEKDFVATGSLGAEYSIDRNWSVRAEYQRYQD MSDDVWDDMDANFFGIGVNYKFAAAPVVAAVVTEEVVEPDPEPVLMSKVHKEEYGTGTFEFDSAKLTESVSERLDNFVSFLNEYPQAQVEITGYTDSSGPAAYNQKLSERRAQAVADYITGAGIDADRLTVKGMGEENPVADNSTREGREKNRRVEVVVPSFEYQEMVQP

[0049] (2) Preparation method: Gene amplification to construct recombinant plasmid, cloning and expression, protein purification.

[0050] (3) Gene amplification and construction of recombinant plasmids: PCR amplification was performed using specific primers for the outer membrane protein OmpA (VPA1186) of Vibrio parahaemolyticus CICC21618 to construct recombinant plasmids.

[0051] (4) Transformation and cloning: The ligation product is transformed into competent cells and induced to express.

[0052] (5) Protein purification: Nitrogen column purification and elution with gradient concentration imidazole elution buffer.

[0053] II. Preparation of Antigens: Monoclonal Antibodies and Paired Antibodies for Specific Vibrio parahaemolyticus

[0054] (1) Experimental animals: Six 6-8 week old BALB / c mice were selected for immunization;

[0055] (2) Emulsification: The purified soluble recombinant OmpA protein was emulsified with an equal amount of complete or incomplete Freund's adjuvant, and mice were injected subcutaneously at multiple points after complete emulsification.

[0056] (3) Immunization: The purified soluble recombinant OmpA protein was emulsified with Freund's complete adjuvant (volume ratio 1:1) to prepare primary immunization emulsions, which were administered to the dorsal region of each mouse via multiple subcutaneous injections. Two booster immunizations were administered with Freund's incomplete adjuvant, with each administration three weeks apart. The antigen doses for the first, second, and third injections were 80 µg, 80 µg, and 40 µg per mouse, respectively.

[0057] (4) Blood collection: Blood was collected from the tail one week after the third immunization. The antiserum titer was determined by indirect non-competitive enzyme-linked immunosorbent assay (ELISA). The concentration of the inactivated Vibrio parahaemolyticus culture was adjusted to 10 using carbonate buffer (pH 9.6). 8 CFU / mL was used to coat 96-well microplates. Serum was serially diluted (starting concentration 1:1000, three-fold gradient) and incubated at 37°C for 30 minutes. Detection was performed using horseradish peroxidase (HRP)-labeled goat anti-mouse IgG (dilution 1:6000). Serum titer was defined as the ability to produce OD. 450 The titer exceeded the highest dilution of the negative control by 2.1 times. After the required titer was achieved, a sprint immunization was performed; fusion was performed 3 days after the immunization by collecting blood from the orbital cavity.

[0058] (5) Fusion and screening: Hybridoma technology was used for fusion, indirect ELISA was used to screen positive cell wells, and limiting dilution method was used to subclone positive wells;

[0059] Cell fusion: Spleen cells were fused with mouse myeloma cells Sp2 / 0 using polyethylene glycol (PEG)-mediated hybridoma technology. Prior to fusion, SP2 / 0 cells were resuscitated and cultured in a CO2 incubator on 1640 medium containing 10% fetal bovine serum to adjust them to the logarithmic growth phase. On the third day after sprint immunization, two mice with the highest antibody titers against different Vibrio parahaemolyticus strains were euthanized, and their spleens were removed. Spleen cells were obtained by washing with 1640 medium, centrifuging, resuspending, and adjusting the concentration for later use. The ratio of spleen cells to SP2 / 0 tumor cells was 8:1.

[0060] Hybridoma cell screening: Hybridoma supernatant was screened for positive results by OmpA, Vibrio parahaemolyticus, and Vibrio strains, and negatively screened for non-Vibrio strains by indirect ELISA. Positive clones were subcloned through three rounds of extreme dilution to obtain stable monoclonal hybridoma cell lines, which were then cryopreserved in liquid nitrogen.

[0061] Preparation of ascites fluid: Ascites fluid was prepared using an in vivo induction method. Healthy female mice were selected at a ratio of 2 mice per cell line. First, sterile, room-temperature paraffin oil was injected. One week later, the prepared hybridoma cells were injected. After 7-8 days of feeding, ascites fluid was extracted, centrifuged, and the supernatant was aliquoted and stored.

[0062] (6) Antibody purification and preservation: Protein A was used to purify ascites fluid, and monoclonal antibodies were obtained after dialysis. The concentration was determined by micro-ultraviolet method, and the antibodies were aliquoted and stored at -20℃.

[0063] (7) Antibody labeling and pairing: The specific monoclonal antibody obtained by the sodium periodate method was coupled with horseradish peroxidase for antibody labeling. The checkerboard method was used for one-to-one pairing. Antibody pairs of Vibrio parahaemolyticus that meet the positive / negative absorbance ratio (P / N) ≥20 were selected for subsequent application screening.

[0064] (8) Antibody pairing screening and confirmation of hybridoma cell lines: Through antibody pairing screening, the optimal pairing was finally determined to be capture antibody 6D11 and detection antibody 12B1. These were obtained from ascites secreted by hybridoma cell lines PTD and TBZ, respectively, after purification. Two hybridoma cell lines were finally identified and named hybridoma cell line PTD and hybridoma cell line TBZ, respectively. They are deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession numbers CGMCC No. 46748 and CGMCC No. 46749, respectively.

[0065] II. Antibody titer determination procedure:

[0066] (1) Dilute the coating buffer with the coating original and coat the 96-well microplate serially, 100 μL / well, incubate at 37°C for 2 hours, remove the microplate and shake it dry, inject 200 μL of PBST solution into each well, shake on a shaker for 3 minutes, shake off the washing solution, pat dry on absorbent paper, and continue washing twice. The following washing methods are the same.

[0067] (2) After thorough washing, block the microplate with blocking buffer, 200 μL / well, incubate at 37°C for 2 hours, then remove and dry for later use;

[0068] (3) Add the serially diluted positive serum to the first 7 rows and columns of the microplate, and add negative serum to the 8th row, 100 μL / well. Incubate at 37°C for 35 minutes, then wash and pat dry.

[0069] (4) Add 100 μL of HRP-labeled goat anti-mouse IgG diluted 1:4000 to each well, incubate at 37°C for 35 minutes, wash four times, and pat dry;

[0070] (5) Add 100 μL of colorimetric solution (TMB to substrate solution ratio is 1:5) to each well, react at 37℃ in the dark for 15 min, remove and add 50 μL of stop solution (2 mol / L sulfuric acid) to each well, and measure the absorbance A450 with an enzyme-linked immunosorbent assay (ELISA) reader.

[0071] III. Labeling of Monoclonal Antibody 12B1

[0072] (1) Add 200 μL of 60mM NaIO4 solution (ultrapure water) dropwise to 200 μL of 10 mg / mL HRP solution, and react at 4°C in the dark for 30 minutes with magnetic stirring.

[0073] 2) While stirring, add 200 μL of 160 mM ethylene glycol solution and react at room temperature in the dark for 30 minutes.

[0074] 3) Add 2 mg of monoclonal antibody while stirring, adjust the pH of the solution to 9.0 with 0.05 M, pH 10.0 CBS, and react at 4°C in the dark for 20 hours.

[0075] 4) Quickly add 80 μL of 5 mg / mL NaBH4 solution, mix well, and let stand at 4°C in the dark for 2 hours.

[0076] 5) Slowly add an equal volume of saturated ammonium sulfate solution, mix well, and let stand at 4°C in the dark for 30 minutes.

[0077] 6) After completion, centrifuge at 5000 rpm for 10 minutes, discard the supernatant, resuspend the precipitate in 500 μL of 10 mM PBS, and dialyze against 10 mM PBS. Cover the beaker with newspaper or similar material to protect it from light, and change the medium 3 times. After dialysis, mix the labeled antibody with an equal volume of glycerol, aliquot and label, and store at -20℃ protected from light. The concentration of the enzyme-labeled antibody is approximately 2 mg / mL.

[0078] IV. Preparation of colloidal gold test strips:

[0079] (1) Synthesis of gold nanoparticles by sodium citrate reduction method: The chloroauric acid solution was heated to 300°C under magnetic stirring and kept boiling for 10 minutes. 1.6 mL of trisodium citrate solution was quickly added and boiling was continued until the color of the solution first turned dark black and finally remained wine red. After cooling to room temperature, it was stored at 4°C.

[0080] (2) Preparation of gold-labeled antibody: K2CO3 solution was added to colloidal gold solution to adjust the pH to 7.5-8, followed by the addition of monoclonal antibody 12B1, and the reaction was carried out at room temperature for 2 hours. Then, BSA solution was added dropwise to the mixture, and the reaction was carried out at room temperature with shaking for 2 hours to block excess sites on the gold nanoparticles. Finally, the mixture was centrifuged at 6000g at 4℃ for 30 min to remove the unconjugated monoclonal antibody 12B1 from the supernatant, and the gold-labeled antibody was resuspended in PBS and stored at 4℃.

[0081] (3) Assembly of colloidal gold test strips: The goat anti-mouse antibody and capture antibody were sprayed onto the NC membrane by the spraying device as C line and T line, respectively. The NC membrane and sample pad were placed in a 37°C oven for 4 hours. Then the NC membrane, sample pad and absorbent pad were cut into 3 mm wide test strips and assembled on a polyvinyl chloride backing card.

[0082] V. Procedure for the determination of Vibrio parahaemolyticus specific double antibody sandwich method:

[0083] a. Coating: Coat the microplate with 4 μg / mL 2G12, 100 μL / well, and incubate at 37℃ for 2 hours;

[0084] b. Washing: Wash the plate three times with PBST for 3 minutes each time, 200 μL / well, and then spin dry the reaction plate;

[0085] c. Sealing: CBS containing 0.2% gelatin, 200 μL / well, sealed at 37℃ for 2 hours;

[0086] d. Washing: Wash the plate three times with PBST for 3 minutes each time, 200 μL / well, and then spin dry the reaction plate;

[0087] e. Samples: Dilute Vibrio parahaemolyticus bacterial suspensions with standard diluents at 3-fold gradients from 10... 7Serially dilute to 13700 CFU / mL, and include a PBS blank control containing 1% Triton X-100. Add 100 μL of sample to each well and incubate at 37°C for 1 hour.

[0088] f. Washing: Wash the plate three times with PBST for 3 minutes each time, 200 μL / well, and then spin dry the reaction plate.

[0089] h. Washing: Wash the plate four times;

[0090] i. Color development: Add 100 μL of color development solution (TMB to substrate solution ratio is 1:5) per well and develop color for 12 minutes;

[0091] j. Termination: Add 50 μL of termination solution per well;

[0092] k. Measurement: OD450 nm was detected using an enzyme-linked immunosorbent assay (ELISA) reader.

[0093] Crossover rate determination: Culture media of other test strains were prepared in a 3-fold gradient from 3 × 10⁻⁶ to 3 × 10⁻⁶. 8 CFU / mL serially diluted to 3.3×10 7 CFU / mL series concentrations were measured using a double-antibody sandwich assay, with blank wells included. Each concentration was analyzed six times, and the average value was calculated. The experiment was repeated three times. The results are as follows:

[0094] 1. Limit of Detection: The antigen concentration corresponding to 2.1 times the average absorbance of the blank is [missing value]. The limit of detection for this Vibrio parahaemolyticus double-antibody sandwich ELISA method for 9 test strains of Vibrio parahaemolyticus is 1.37 × 10⁻⁶. 4 ~ 10 6 CFU / mL.

[0095] 2. Cross-reactivity: Among the 29 test strains, only 10 showed cross-reactivity. 8 The boiled Vibrio parahaemolyticus solution at concentrations above CFU / mL showed cross-reactivity with Vibrio parahaemolyticus, while no cross-reactivity was observed with different concentrations of boiled bacterial solutions for all other 27 test strains. This indicates that the established double-antibody sandwich ELISA method has good specificity for Vibrio parahaemolyticus.

[0096] V. Food Sample Testing

[0097] Twenty-two seafood samples were analyzed using the established DAS-ELISA method, culture-based thiosulfate citrate bile salt sucrose agar (TCBS agar), and PCR.

[0098] Each 25-gram sample was homogenized in 225 mL of 3% sodium chloride alkaline peptone water (APW) and enriched at 36±1 °C for 8–18 hours.

[0099] (1) Direct detection of Vibrio parahaemolyticus in seafood: 25g of each seafood sample was added to a sterile homogenizing bag, and 225ml of 3% sodium chloride alkaline buffered peptone water (APW) was added. The mixture was homogenized in a sterile homogenizer for 2 minutes to prepare a 1:10 sample homogenate. ELISA was used for identification, and PCR and TCBS plate culture were used for confirmation.

[0100] (2) Determination of artificially contaminated samples: Shrimp and squid samples were artificially contaminated with 10 CFU / g of standard VP21619 and enriched in 3% sodium chloride APW at 37°C for 0, 1, 2, 3, 4, 5, 6, 8, 10, 12, and 18 hours (PBS as a negative control). The boiled enrichment at each time point was detected using a developed DAS-ELISA to determine detectability. Subsequently, based on the DAS-ELISA results, key time points (0, 6, 12, and 18 hours) were selected to evaluate the optimal enrichment time for LFIA.

[0101] The experimental results are as follows: The limit of detection for Vibrio parahaemolyticus in seafood using the double-antibody sandwich ELISA method is 10. 5 The detection limit for Vibrio parahaemolyticus in seafood was 10 CFU / mL (without enrichment) after 6 hours of enrichment (no enrichment). The results were consistent with those of the selective plate assay using the national standard method. The results are shown in Tables 1 and 2. Figure 5 As shown:

[0102]

[0103] Table 1. Results of double-antibody sandwich immunoassay and TCBS plate culture for Vibrio halophilus-specific gene PCR detection in commercially available fresh / processed seafood samples: +++, strong positive result (OD450 ≥ 1.6); ++, moderately positive result (1.0 ≤ OD450 < 1.6); +, weak positive result (0.4 ≤ OD450 < 1.0); -, negative result (OD450 < 0.4). The background value of APW enrichment broth was 0.151 ± 0.02.

[0104]

[0105] Table 2 ( Figure 5 The results of ELISA and colloidal gold test strips on artificially contaminated samples showed that squid and shrimp with a contamination concentration of 10 CFU / ml could be detected by ELISA and colloidal gold test strips after only 6 hours of enrichment, which greatly shortened the time required for actual sample testing.

[0106] The above descriptions are all preferred embodiments of the present invention. For those skilled in the art, any modifications to the present invention in various equivalent forms without departing from the principle of the present invention shall fall within the protection scope of the appended claims.

Claims

1. A hybridoma cell line combination containing a monoclonal antibody against Vibrio parahaemolyticus OmpA protein, characterized in that: The hybridoma cell line combination consists of hybridoma cell line PTD and hybridoma cell line TBZ; Among them, hybridoma cell line PTD was deposited at the China General Microbiological Culture Collection Center on November 13, 2025, with accession number CGMCC No. 46748, and hybridoma cell line TBZ was deposited at the China General Microbiological Culture Collection Center on November 13, 2025, with accession number CGMCC No. 46749.

2. A monoclonal antibody combination against Vibrio parahaemolyticus OmpA protein, characterized in that: The monoclonal antibody combination consists of monoclonal antibody 6D11 and monoclonal antibody 12B1 secreted by the hybridoma cell line combination described in claim 1.

3. The use of the monoclonal antibody combination according to claim 2 in the preparation of a kit, reagent, or test strip for detecting Vibrio parahaemolyticus.

4. A double-antibody sandwich ELISA kit for detecting Vibrio parahaemolyticus, characterized in that: The kit comprises the monoclonal antibody combination of claim 2, wherein monoclonal antibody 6D11 is used as the coating antibody and monoclonal antibody 12B1 is used as the detection antibody.

5. The reagent kit according to claim 4, characterized in that: The kit also includes standard solutions, blocking solutions, diluents, washing solutions, colorimetric solutions, and stop solutions.

6. A colloidal gold immunochromatographic test strip for detecting Vibrio parahaemolyticus, characterized in that: The test strips were constructed as follows: Gold nanoparticles were conjugated with monoclonal antibody 12B1 for binding to the antigen. Monoclonal antibody 6D11 and goat anti-mouse IgG antibody were diluted with 0.01 mol / L phosphate buffer and sprayed onto a nitrocellulose membrane at a spraying rate of 0.9 µL / cm to form the detection line (T line) and control line (C line), respectively. The NC membrane and sample pad were placed in a 37°C oven for 4 hours. Subsequently, the NC membrane, sample pad, and absorbent pad were cut into 3 mm wide test strips and assembled onto a polyvinyl chloride backing card.

7. A rapid double-antibody sandwich ELISA method for detecting Vibrio parahaemolyticus, characterized in that: S1: Preparation of specific Vibrio parahaemolyticus monoclonal antibody: Using purified soluble recombinant OmpA protein as an immunogen, 6-8 week old BALB / c mice were immunized. After immunization and fusion, cells strongly positive for Vibrio parahaemolyticus were screened for subcloning to obtain Vibrio parahaemolyticus specific monoclonal antibody. S2: Screening for paired monoclonal antibodies: Purify the Vibrio parahaemolyticus-specific monoclonal antibodies obtained in step S1, and label them with horseradish peroxidase (HRP). After successful labeling, perform sandwich pairing. S3: Establishment of a sandwich ELISA method specific to Vibrio parahaemolyticus: The paired monoclonal antibodies obtained in step S2 are used, with monoclonal antibody 6D11 as the coating antibody and monoclonal antibody 12B1 as the detection antibody. The ELISA plate is coated with monoclonal antibody 6D11 to specifically capture Vibrio parahaemolyticus. The enzyme-labeled antibody 12B1-HRP on the ELISA plate binds to the captured Vibrio parahaemolyticus. After the substrate is added, it is catalyzed by HRP enzyme and produces an absorbance at 450 nm. The result is determined according to the P / N value. Specifically: if Vibrio parahaemolyticus is captured by the coating antibody 6D11 and binds to the enzyme-labeled antibody 12B1-HRP, and catalyzes the substrate to produce an absorbance at 450 nm (P / N ≥ 2.1), it is judged as positive; if the concentration of Vibrio parahaemolyticus is too low (P / N < 2.1), it is judged as negative.

8. The rapid detection method for Vibrio parahaemolyticus using a double-antibody sandwich ELISA according to claim 7, characterized in that: The specific detection steps in S3 are as follows: (1) Coating: Coat the microplate with 4 μg / mL 6D11, using 100 μL / well, and incubate at 37℃ for 2 h; (2) Washing: Wash the plate three times with PBST for 3 minutes each time, using 200 μL / well, and then spin dry the reaction plate; (3) Sealing: Seal the wells of the plate with a carbonate buffer solution containing 0.2% gelatin, at a rate of 200 μL / well, and seal at 37°C for 2 hours; (4) Washing: Same as step (2); (5) Samples: Vibrio parahaemolyticus bacterial suspension was diluted with PBS containing 1% Triton X-100 at a 3-fold gradient from 10... 7 CFU / mL was serially diluted to 13700 CFU / mL. A blank control containing 1% Triton X-100 in PBS was also set up. 100 μL of sample was added to each well and incubated at 37°C for 1 hour. (6) Washing: Same as step (2); (7) Add 4 μg / mL enzyme-labeled antibody 12B1-HRP, 100 μL / well, and react at 37℃ for 1 h; (8) Washing: Wash the board four times; (9) Color development: Add color development solution at a ratio of 1:5 of TMB to substrate solution, with a volume of 100 μL / well, and develop color for 12 min; (10) Termination: Add 50 μL of termination solution per well; (11) Measurement: OD450nm was measured using an enzyme-linked immunosorbent assay (ELISA) reader.

9. A rapid double-antibody sandwich ELISA method for detecting Vibrio parahaemolyticus according to claim 7 or 8, characterized in that: The pairing parameters included 4 μg / mL of coated antibody 6D11, coating buffer (pH 9.6, 0.01 M carbonate buffer), washing buffer (0.5% Tween 20 solution prepared with PBS), and a standard concentration of 10 μg / mL. 7 The standard was diluted with 1% Triton X-100 solution at pH 7.2 using 0.01M PBS, and the enzyme-labeled antibody 12B1-HRP concentration was 4 μg / mL.

10. A rapid double-antibody sandwich ELISA method for detecting Vibrio parahaemolyticus according to claim 7 or 8, characterized in that: The standard samples include six Vibrio parahaemolyticus standard strains CICC 21618, CICC 21619, CICC 21528, CICC10552, CGMCC 1.1616, and CICC 21617, as well as three Vibrio parahaemolyticus isolates.

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