Monoclonal antibody and kit specifically binding to serum type 7 antigen of Actinobacillus pleuropneumoniae

A technology of monoclonal antibody and pleuropneumonia, applied in the field of biotechnology and animal quarantine, to achieve the effect of easy quality and mass production

Inactive Publication Date: 2016-06-22
SHANGHAI ENTRY EXIT INSPECTION & QUARANTINE BUREAU OF P R C +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There is also no kit for detecting App antibodies by competitive ELISA

Method used

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  • Monoclonal antibody and kit specifically binding to serum type 7 antigen of Actinobacillus pleuropneumoniae
  • Monoclonal antibody and kit specifically binding to serum type 7 antigen of Actinobacillus pleuropneumoniae
  • Monoclonal antibody and kit specifically binding to serum type 7 antigen of Actinobacillus pleuropneumoniae

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Embodiment 1 Hybridoma cell line preparation and the preparation of monoclonal antibody

[0050] Step 1. Preparation of immunogen: prepare chocolate medium, streak on the chocolate medium to revive Actinobacillus pleuropneumoniae serotype 7 standard strain (AppS7), mass culture, wash the bacteria with normal saline, 8000r / min Centrifuge for 10 minutes, discard the supernatant, suspend the bacterial pellet with normal saline, centrifuge, and wash the bacteria three times in this way. The bacterial pellet was suspended in saline containing 0.5% formaldehyde, placed at 4°C overnight to inactivate, centrifuged at 8000r / min for 10min to remove formaldehyde, and washed 3 times with saline. The whole antigen of AppS7 bacteria suspended in normal saline is used as the immunogen; step 2, BALB / C mouse immunization: the above-mentioned immunogen (containing 10 4 Actinobacillus pleuropneumoniae) was emulsified with an equal volume of Freund's complete adjuvant, and immunized by in...

Embodiment 2

[0053] Example 2 Chemically labeled monoclonal antibody in vitro

[0054] The main steps are:

[0055] 1. Subtype identification of monoclonal antibody: use the mouse monoclonal antibody detection kit (MouseMabIsotyingtestkit) produced by HyCultBiotechnology in the Netherlands, and the steps are as follows:

[0056] a) Properly dilute the cell supernatant or ascites with pH 7.4 PBS to an antibody concentration of 1-50 μg / mL for later use;

[0057] b) Add 500 μL of diluent to the test tube containing a test strip;

[0058] c) Add the diluted sample in 500 μL) to the detection tube;

[0059] d) Submerge the test strip completely and stir gently;

[0060] e) Gently shake the bottle to mix the rabbit anti-mouse κ chain micelles, and add 1 mL to the detection tube;

[0061] f) Shake in an environment of 18-25°C for about 30 minutes to interpret the results, and the test strip is always immersed in the solution during the shaking process.

[0062] After testing, the monoclonal ...

Embodiment 3

[0083] Example 3 Competitive ELISA detects Actinobacillus pleuropneumoniae serum type 7 antibody kit test

[0084] The test procedure of the competition ELISA kit includes the following contents: 1) Antigen preparation 2) Antigen titer titration 3) Determination of optimal dilution concentration of sample serum 4) Determination of CUTOFF value 5) Competition ELISA test steps a) Coating antigen, 4°C refrigerator overnight; b) Wash the plate 3 times; c) Block at 37°C for 1 hour; d) Wash the plate 3 times; e) Add the tested serum and add horseradish peroxidase-labeled anti-App serum type 7 antigen For monoclonal antibody, react at 37°C for 30 min; f) wash the plate 3 times; g) add substrate, react at 37°C for 10 min; h) terminate the reaction; i) detect the absorbance value (OD value); j) calculate and judge the result. details as follows:

[0085] 1) Antigen preparation

[0086] Bacterial colonies cultured on TSCA medium (containing 0.1% NAD) for 6 hours were eluted with norma...

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Abstract

The invention discloses a monoclonal antibody capable of specifically combining an actinobacillus pleuropneumoniae serum 7-type antigen and a hybridoma cell line for secreting a monoclonal antibody. The hybridoma cell line is preserved in China General Microbiological Culture Collection Center with the preservation number of CGMCC No.9051. The invention also discloses a preparation method of the monoclonal antibody. The invention also discloses the monoclonal antibody subjected to in-vitro chemical marking treatment. The invention also discloses a kit for detecting the actinobacillus pleuropneumoniae serum 7-type antigen by adopting competitive ELISA. The competitive ELISA kit can be used for detecting a sample within one hour, is higher than a present commercial indirect ELISA detection kit by 0.5 hour in detection speed, is higher than blocking ELISA used in the inspection and quarantine industry standard by 1.5 hours in detection speed, can detect multiple batches of samples within one day, and can meet the rapid epidemic disease diagnosis requirement.

Description

technical field [0001] The invention belongs to the technical field of biotechnology and animal quarantine, and specifically relates to a monoclonal antibody specifically binding to the serum type 7 antigen of Actinobacillus pleuropneumoniae and a hybridoma cell line secreting it. In addition, the invention also relates to a competitive ELISA detection Actinobacillus pleuropneumoniae serotype 7 antibody kit. Background technique [0002] Porcine contagious pleuropneumoniae is a respiratory infectious disease of pigs caused by Actinobacillus pleuropneumoniae (App for short). The mortality rate of the most acute type can be as high as 80-100%, and the chronic type can cause the pig to grow slowly and become a dead pig. This disease is one of the common pig diseases in intensive pig farms and is imported from my country. One of the swine diseases of boar quarantine. my country has repeatedly detected porcine infectious pleuropneumonia from imported breeding pigs from the United...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/12
CPCG01N33/577
Inventor 李树清陈志飞张强王巧全林颖峥刘雨潇吴建详陆承平宋青唐智芳
Owner SHANGHAI ENTRY EXIT INSPECTION & QUARANTINE BUREAU OF P R C
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