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CN-122012404-A - Double-antibody sandwich ELISA kit for rapidly detecting vibrio parahaemolyticus and application thereof

CN122012404ACN 122012404 ACN122012404 ACN 122012404ACN-122012404-A

Abstract

The invention discloses a double-antibody sandwich ELISA kit for rapidly detecting vibrio parahaemolyticus and application thereof, wherein optimal pairing 6D11 and 12B1 are obtained through preparation of monoclonal antibodies and screening of different antibody pairings, and a double-antibody sandwich ELISA method for detecting vibrio parahaemolyticus is established by taking monoclonal antibody 6D11 as a coating antibody and taking 12B1-HRP as a detection antibody. The method has high sensitivity (1.37X10 4 CFU/mL) and good specificity to the vibrio parahaemolyticus, and has cross reaction to the 9 tested vibrio parahaemolyticus. And the method has no cross reaction (27/29) to other common 27 vibrio internal and external bacteria, the detection limit of the vibrio parahaemolyticus in the seafood after 6 hours of enrichment is 10 CFU/mL, and the detection result is consistent with the detection result of a national standard method selective flat plate. The method provides an accurate, reliable and quick analysis means for detecting the vibrio parahaemolyticus in the marine products.

Inventors

  • WANG WENBIN
  • ZHU WENTAO
  • LIU LIQIANG
  • QIU JUNJIE
  • LI WEN
  • DONG XIAOYU
  • XU ZHUO
  • PAN SAIKUN

Assignees

  • 江苏海洋大学

Dates

Publication Date
20260512
Application Date
20260104

Claims (10)

  1. 1. A hybridoma cell strain combination of an anti-vibrio parahaemolyticus OmpA protein monoclonal antibody is characterized in that the hybridoma cell strain combination consists of a hybridoma cell strain PTD and a hybridoma cell strain TBZ; Wherein, the hybridoma cell strain PTD is preserved in China general microbiological culture collection center (CGMCC No. 46748) at the year of 2025 and 11 and 13, and the hybridoma cell strain TBZ is preserved in China general microbiological culture collection center (CGMCC No. 46749) at the year of 2025 and 11 and 13.
  2. 2. A monoclonal antibody combination of the vibrio parahaemolyticus OmpA protein, which is characterized by consisting of a monoclonal antibody 6D11 and a monoclonal antibody 12B1 secreted by the hybridoma cell line combination of claim 1.
  3. 3. The use of a monoclonal antibody combination according to claim 2 for the preparation of a kit or reagent or test strip for the detection of vibrio parahaemolyticus.
  4. 4. A double-antibody sandwich ELISA kit for detecting vibrio parahaemolyticus is characterized in that the kit comprises the monoclonal antibody combination of claim 2, wherein monoclonal antibody 6D11 is used as a coating antibody, and monoclonal antibody 12B1 is used as a detection antibody.
  5. 5. The kit according to claim 4, wherein the kit further comprises a standard solution, a blocking solution, a diluent, a washing solution, a color development solution and a stop solution.
  6. 6. A colloidal gold immunochromatographic test strip for detecting vibrio parahaemolyticus is characterized by comprising the following steps of coupling gold nanoparticles with a monoclonal antibody 12B1 for antigen binding, diluting a monoclonal antibody 6D11 and a goat anti-mouse IgG antibody with 0.01 mol/L phosphate buffer solution, spraying the diluted monoclonal antibody and goat anti-mouse IgG antibody on a nitrocellulose membrane at a spraying rate of 0.9 mu L/cm to form a detection line, namely a T line and a quality control line, namely a C line, respectively, placing an NC membrane and a sample pad in a 37 ℃ oven for 4 hours, cutting the NC membrane, the sample pad and the absorption pad into test strips with a width of 3 mm, and assembling the test strips on a polyvinyl chloride backing card.
  7. 7. A double-antibody sandwich ELISA method for rapidly detecting vibrio parahaemolyticus is characterized in that: S1, preparing a specific vibrio parahaemolyticus monoclonal antibody, namely immunizing a BALB/c mouse with 6-8 weeks old by taking purified soluble recombinant OmpA protein as an immunogen, and screening to subclone cells with strong positive vibrio parahaemolyticus by immunization and fusion to obtain the vibrio parahaemolyticus specific monoclonal antibody; s2, screening a pairing monoclonal antibody, namely purifying the vibrio parahaemolyticus specific monoclonal antibody obtained in the step S1, respectively marking horseradish peroxidase HRP, and carrying out sandwich method pairing after the identification marking is successful; S3, establishing a vibrio parahaemolyticus specific sandwich ELISA method, namely using the paired monoclonal antibody obtained in the step S2 as a coating antibody, using a monoclonal antibody 6D11 as a detection antibody, using a monoclonal antibody 12B1 as a detection antibody, using an ELISA plate coated monoclonal antibody 6D11 for specifically capturing vibrio parahaemolyticus, combining the ELISA plate coated monoclonal antibody 12B1-HRP with the captured vibrio parahaemolyticus, adding a substrate, catalyzing the substrate by HRP enzyme and generating an absorption value at 450nm, and judging the result according to the P/N value, wherein if the vibrio parahaemolyticus is captured by the coated antibody 6D11 and combined with the ELISA plate 12B1-HRP, catalyzing the substrate to generate an absorption value P/N at 450nm is more than or equal to 2.1, judging positive, and if the concentration of the vibrio parahaemolyticus is too low, P/N is less than 2.1 and judging negative.
  8. 8. The double-antibody sandwich ELISA method for rapidly detecting vibrio parahaemolyticus according to claim 7, wherein the specific detection steps in S3 are as follows: (1) Coating the ELISA plate with 4. Mu.g/mL of 6D11 at a dose of 100. Mu.L/well and incubating for 2h at 37 ℃; (2) Washing, namely washing the plate three times by using PBST (Poly Butylene terephthalate) for 3min each time with the dosage of 200 mu L/hole, and then spin-drying the reaction plate; (3) Blocking, namely blocking the plate holes by using a carbonate buffer solution containing 0.2% gelatin, wherein the dosage of the carbonate buffer solution is 200 mu L/hole, and blocking for 2 hours at 37 ℃; (4) Washing, wherein the washing is the same as the step (2); (5) Sample, namely continuously diluting the vibrio parahaemolyticus bacterial liquid from 10 7 CFU/mL to 13700CFU/mL by using PBS containing 1% Triton X-100, adding 100 mu L of sample into each hole, and incubating lh at 37 ℃ by using PBS containing 1% Triton X-100; (6) Washing, wherein the washing is the same as the step (2); (7) Adding 4 mug/mL enzyme-labeled antibody 12B1-HRP, using 100 mug/hole, and reacting for 1h at 37 ℃; (8) Washing the plate four times; (9) Color development, namely adding a color developing solution according to the proportion of TMB to substrate solution of 1:5, and developing for 12min with the dosage of 100 mu L/hole; (10) Stopping, namely adding 50 mu L/hole of stopping solution; (11) The OD450nm was measured by an enzyme-labeled instrument.
  9. 9. The double-antibody sandwich ELISA method for rapidly detecting vibrio parahaemolyticus according to claim 7 or 8, wherein the pairing parameters comprise 4 mug/mL of coated antibody 6D11, the coating solution is pH9.6 and 0.01M carbonate buffer solution, the washing solution is Tween 20 solution with concentration of 0.5% prepared by PBS, the standard concentration is 10 7 CFU/mL, the standard diluent is pH7.2 and 1% Triton X-100 solution with concentration prepared by 0.01M PBS, and the enzyme-labeled antibody 12B1-HRP concentration is 4 mug/mL.
  10. 10. The double-antibody sandwich ELISA method for rapidly detecting vibrio parahaemolyticus according to claim 7 or 8, wherein the standard comprises six standard strains of vibrio parahaemolyticus CICC 21618, CICC 21619, CICC 21528, CICC 10552, CGMCC 1.1616, CICC 21617 and three isolated strains of vibrio parahaemolyticus.

Description

Double-antibody sandwich ELISA kit for rapidly detecting vibrio parahaemolyticus and application thereof Technical Field The invention belongs to the technical field of immunoassay, and particularly relates to a double-antibody sandwich ELISA kit for rapidly detecting vibrio parahaemolyticus and application thereof. Background Vibrio parahaemolyticus (V. Parahaemeolyticus) is a gram-negative, halophilic zoonotic bacterium which is widely distributed in marine organisms such as fish, shrimp and shellfish, and is associated with acute hepatopancreatic necrosis and has caused great loss to the world-wide shrimp culture. Vibrio parahaemolyticus is also one of the main causative agents of food-borne diseases, and places a heavy burden on public health worldwide. In particular, consumption of uncooked seafood or cross-contaminated foods may cause acute gastroenteritis and in severe cases even sepsis. Thermostable Direct Hemolysin (TDH) and TDH-related hemolysin (TRH) are core virulence factors inducing the Kanagawa phenomenon, while thermostable direct hemolysin (TLH) is not directly pathogenic, but is preserved in most strains, and the three genes are used as detection markers of vibrio parahaemolyticus. In China, a total of 1,772 outbreaks of epidemic conditions involving Vibrio parahaemolyticus, and more than 27,000 cases, were reported during 2010 to 2020. Other high-burden areas, including japan and southeast asia, continue to report localized outbreaks. The limit of Vibrio parahaemolyticus in food in China is regulated to be 5 samples, the content of Vibrio parahaemolyticus in1 sample is allowed to be 100 MPN/g to 1000 MPN/g at most, and other samples are required to be less than or equal to 100 MPN/g. Any sample must not be higher than 1000 MPN/g. Traditional methods for detecting Vibrio parahaemolyticus, including culture-based methods and nucleic acid-based detection techniques, have limitations in terms of time efficiency, ease of operation, detection throughput, and field applicability. Immunological detection methods such as enzyme-linked immunosorbent assay (ELISA) and Lateral Flow Immunochromatography (LFIA) have the advantages of being rapid, low in cost, and small in dependence on equipment, and have been successfully applied to the detection of other food-borne pathogens. However, due to the lack of high abundance of species-specific surface antigens, coupled with the interference of complex seafood matrices, immunodetection remains a challenge in terms of sensitivity and specificity, resulting in fewer mature products worldwide. The existing immunoassay methods for thermostable direct hemolysin-related hemolysin (TRH), pir toxin or LppQ-87G 48-based detection methods are deficient in comprehensively unbiased detection of all vibrio parahaemolyticus serotypes or in achieving low detection Limit (LOD). The immunomagnetic bead separation-LFIA system based on commercial polyclonal antibodies and monoclonal antibodies (mabs) has been reported in the past, with lower detection limits (10 CFU/mL) reported after the enrichment step, but these methods did not evaluate the specificity of the antibodies and the overall specificity of the method. Thus, there is an urgent need for a rapid, highly sensitive and highly specific immunoassay method for vibrio parahaemolyticus. Outer membrane protein A (OmpA) of Vibrio parahaemolyticus is the main outer membrane protein thereof, and is involved in osmotic pressure regulation. The protein is highly expressed, and can enhance the drug resistance of bacteria to antibiotics by regulating the permeability of small molecules, so that the protein is regarded as a potential subunit vaccine candidate molecule. Comparative genomics and surface proteomics analyses of the earlier stage of the subject group showed that 3 OmpA proteins have a level conservation, while retaining sequence characteristics specific to vibrio parahaemolyticus, suggesting that they can be used as reliable immunodiagnostic markers. Notably, ompA is highly exposed to bacterial surfaces under a variety of environmental conditions and is regulated by natural antibacterial agents. Immunological studies prove that the vibrio parahaemolyticus recombinant OmpA can induce strong humoral immune response and partial protective immunity in mice, and the reported protection rate is as high as 73%. In addition, 3 multivalent vaccine candidate molecules are identified in the heterozygous OmpA obtained by screening by a DNA shuffling technology, and can effectively resist extracellular infection of vibrio alginolyticus (V. alginolyticus) and intracellular infection of Edwardsiella tarda (EDWARDSIELLA TARDA). Since OmpA has stable surface exposure, high abundance, strong immunogenicity and conservation, it is an ideal candidate molecule for developing vibrio parahaemolyticus sensitivity and specific immunity detection methods. Despite the progress made in the above-described studies, there are still few reports on t