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Vibrio vulnificus flagellin monoclonal antibody and antigen capture ELISA (Enzyme Linked Immunosorbent Assay) kit

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

Active Publication Date: 2013-04-17
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 process of Vibrio vulnificus monoclonal antibodies, inactivated VV cells were often used as antigens, and most of the proteins of the cells were common proteins of the genus, which did not have interspecies specificity, resulting in the harvested monoclonal antibodies Interspecies cross-reactivity within the Vibrio genus is difficult to avoid

Method used

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  • Vibrio vulnificus flagellin monoclonal antibody and antigen capture ELISA (Enzyme Linked Immunosorbent Assay) kit
  • Vibrio vulnificus flagellin monoclonal antibody and antigen capture ELISA (Enzyme Linked Immunosorbent Assay) kit
  • Vibrio vulnificus flagellin monoclonal antibody and antigen capture ELISA (Enzyme Linked Immunosorbent Assay) kit

Examples

Experimental program
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Effect test

Embodiment 1

[0018] Example 1 Preparation of Vibrio vulnificus Flagellin Monoclonal Antibody

[0019] 1. Cell culture and extraction of flagellin

[0020] Vibrio vulnificus (VV) (ATCC 1758, 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 and ce...

Embodiment 2

[0066] Example 2 Vibrio vulnificus flagellin capture ELISA kit

[0067] 1. Composition of Vibrio vulnificus Flagellin Capture ELISA Kit

[0068] The solid phase carrier coated with the polyclonal antibody of Vibrio vulnificus flagellin is the microtiter plate, the monoclonal antibody of Vibrio vulnificus flagellin labeled with horseradish peroxidase, the substrate reaction solution of the enzyme, positive and negative controls, washing solution and reaction termination solution.

[0069] (1) ELISA plate coated with polyantibody

[0070]Vulnificus vulnificus flagellin polyclonal antibody (prepared as in Example 1) was diluted to a concentration of 10 μg / mL with PBS buffer, and coated with 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 plate, and blocked at 37°C for 1...

Embodiment 3

[0083] Example 3 The method of using Vibrio vulnificus flagellin capture ELISA kit

[0084] 1. Processing of samples to be tested

[0085] 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.

[0086] 2. Add control and test samples

[0087] 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 .

[0088] 3. Add enzyme-labeled monoclonal antibody

[0089] 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°C for...

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Abstract

The invention discloses a vibrio vulnificus flagellin monoclonal antibody and an antigen capture ELISA (Enzyme Linked Immunosorbent Assay) kit. The vibrio vulnificus flagellin monoclonal antibody is generated and secreted by a hybridoma cell strain with the preservation number of CGMCC No.6755. The vibrio vulnificus flagellin monoclonal antibody disclosed by the invention can be used for detecting the vibrio vulnificus. The invention also discloses a vibrio vulnificus flagellin capture ELISA kit.

Description

technical field [0001] The invention belongs to the field of immunology, and in particular relates to a vibrio vulnificus flagellin monoclonal antibody, a hybridoma cell line and an antigen capture ELISA kit. Background technique [0002] Vibrio vulnificus (VV) is a halophilic Vibrio that widely exists in seawater and seafood. The human body can be infected by raw seafood or contact with seawater and seafood through broken limb wounds. It is common in fishermen working at sea and sea drillers, causing sepsis and soft tissue infection. This bacteria is very harmful to patients with diabetes, alcoholic liver disease, liver cirrhosis, hepatitis, and liver dysfunction. It can cause Vibrio vulnificus sepsis, with rapid onset, rapid progression and high mortality. Rapid and accurate early diagnosis is a powerful guarantee to guide the correct clinical use of drugs to achieve the desired therapeutic effect; at the same time, the treatment of Vibrio vulnificus is still empirical an...

Claims

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Application Information

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IPC IPC(8): C07K16/12C12N5/20G01N33/68G01N33/577G01N33/569
Inventor 曾静张西萌张蕾魏海燕周熙城刘莉
Owner BEIJING ENTRY EXIT INSPECTION & QUARANTINE BUREAU INSPECTION & QUARANTINE TECH CENT
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