Vibrio parahaemolyticus flagellin monoclonal antibody and antigen capture ELISA (enzyme-linked immunosorbent assay) kit

A hemolytic vibrio and monoclonal antibody technology, applied in the field of immunology, can solve the problems of no cross-species specificity and it is difficult to avoid cross-reactions between vibrio species, and achieve good specificity and stability

Inactive Publication Date: 2012-09-12
BEIJING ENTRY EXIT INSPECTION & QUARANTINE BUREAU INSPECTION & QUARANTINE TECH CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In the previous preparation of monoclonal antibodies against Vibrio parahaemolyticus, inactivated VP cells were often used as antigens, and most of the proteins in the cells were common proteins of the genus, which did not have interspecies specificity, resulting in poor harvest Monoclonal antibodies are difficult to avoid interspecies cross-reactivity within the Vibrio genus

Method used

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  • Vibrio parahaemolyticus flagellin monoclonal antibody and antigen capture ELISA (enzyme-linked immunosorbent assay) kit
  • Vibrio parahaemolyticus flagellin monoclonal antibody and antigen capture ELISA (enzyme-linked immunosorbent assay) kit
  • Vibrio parahaemolyticus flagellin monoclonal antibody and antigen capture ELISA (enzyme-linked immunosorbent assay) kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Example 1 Preparation of Vibrio parahaemolyticus Flagellin Monoclonal Antibody

[0020] 1. Cell culture and extraction of flagellin

[0021] Vibrio parahemolyticus (ATCC 17802, stored in the laboratory) was inoculated in T1N1 medium (purchased from Beijing Land Bridge Technology Co., Ltd., item number CM168), activated at 36°C for 24 hours; a single colony was picked and inoculated in a medium containing 2% NaCl TSA medium (purchased from Beijing Land Bridge Technology Co., Ltd., product number CM417) was grown for 18-24 h; well-grown colonies were evenly spread on the TSA (2% NaCl) medium with a sterile cotton swab, and cultured at 36°C for 18 h. ±2h. The next day, the bacterial cells were scraped off with a sterile cotton swab and suspended in a pre-cooled 0.15M NaCl solution, kept in an ice bath for 15-30 min, homogenized at 1000 rpm for 45 sec, and immediately placed on ice for 10 min. Centrifuge the homogenate at 10,000 rpm for 10 min at 4°C; take the supernatant...

Embodiment 2

[0068] Example 2 Vibrio parahaemolyticus flagellin capture ELISA kit

[0069] 1. Composition of Vibrio parahaemolyticus flagellin capture ELISA kit

[0070] The solid phase carrier that has been coated with the polyclonal antibody of Vibrio parahaemolyticus flagellin is the microtiter plate, the monoclonal antibody of flagellin of Vibrio parahaemolyticus labeled with horseradish peroxidase, the substrate reaction solution of the enzyme, the positive and Negative control, washing solution and reaction stop solution.

[0071] (1) ELISA plate coated with polyantibody

[0072] Dilute the Vibrio parahaemolyticus flagellin polyclonal antibody (prepared as in Example 1) with PBS buffer to a concentration of 10 μg / mL, and coat a 96-well EIA high-efficiency binding microtiter plate at 100 μL / well, overnight at 4°C. After taking it out, wash the plate 3 times with PBST and shake dry. 2% BSA was dissolved in PBS solution as a blocking solution, 100 μL / well was added to the microtiter ...

Embodiment 3

[0085] Example 3 The method of using Vibrio parahaemolyticus flagellin capture ELISA kit

[0086] 1. Processing of samples to be tested

[0087] Take 1 mL of the enrichment solution of the sample to be tested, centrifuge at 10,000 rpm for 2 minutes, discard the supernatant, wash the pellet twice with PBS buffer, and add a small amount of 200 μL PBS to resuspend the pellet. Boiling water bath for 5min.

[0088] 2. Add control and test samples

[0089] Take 100 μL of the sample to be tested and add to the corresponding enzyme-labeled well, positive control and negative control 100 μL / well, tap the side of the sample plate to ensure that the sample evenly covers the bottom of the well, incubate at 37°C for 1 hour, wash the plate with PBST 3 times, and shake dry .

[0090] 3. Add enzyme-labeled monoclonal antibody

[0091] Add 100 μL / well of enzyme-labeled monoclonal antibody working solution (1:1000 dilution of enzyme-labeled monoclonal antibody in PBS buffer), incubate at 37...

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Abstract

The invention discloses a vibrio parahaemolyticus flagellin monoclonal antibody and an antigen capture ELISA (enzyme-linked immunosorbent assay) kit. The vibrio parahaemolyticus flagellin monoclonal antibody is produced by secreting of a hybridoma cell strain with the preservation number of CGMCC (China General Microbiological Culture Collection) No.6061. The vibrio parahaemolyticus flagellin monoclonal antibody can be used for detecting vibrio parahaemolyticus. The invention also discloses a vibrio parahaemolyticus flagellin capture ELISA (enzyme-linked immunosorbent assay) kit.

Description

technical field [0001] The invention belongs to the field of immunology, and in particular relates to a flagellin monoclonal antibody of Vibrio parahaemolyticus, a hybridoma cell line and an antigen capture ELISA kit. Background technique [0002] Vibrio parahemolyticus (VP) is a halophilic bacterium. Vibrio parahaemolyticus food poisoning is caused by eating foods containing the bacteria, mainly from seafood, such as cuttlefish, sea fish, sea shrimp, sea crab, jellyfish, and preserved foods with high salt content, such as pickles, pickled vegetables, etc. meat etc. The bacterium has a strong survival ability and can survive for more than 1 month on rags and cutting boards, and can survive in seawater for 47 days. Clinically, the main symptoms are acute onset, abdominal pain, vomiting, diarrhea and watery stool. The disease mostly occurs in coastal areas in summer and autumn, often causing collective disease. In recent years, due to the air transportation of seafood, the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/12C12N5/20G01N33/68G01N33/577
Inventor 曾静魏海燕张蕾张西萌
Owner BEIJING ENTRY EXIT INSPECTION & QUARANTINE BUREAU INSPECTION & QUARANTINE TECH CENT
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