Kit for screening and detecting African swine fever virus P30 protein monoclonal antibody and preparation method thereof

An African swine fever virus, P30 technology, applied in virus disease diagnosis technology, animal quarantine, and biomedicine fields, can solve the problem of lack of methods and kits, and achieve the effect of improving screening efficiency

Pending Publication Date: 2020-07-31
杭州恒奥科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In order to be able to research and prepare monoclonal antibodies for African swine fever virus detection, we use molecular biology techniques to clone and express P30 protein, and use this as a basis to study monoclonal antibodies against P30 protein, but in large-scale screening and evaluation For the monoclonal antibody against P30 protein, it was found that there is no suitable method and kit, because the conventional method needs to use African swine fever virus for evaluation

Method used

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  • Kit for screening and detecting African swine fever virus P30 protein monoclonal antibody and preparation method thereof
  • Kit for screening and detecting African swine fever virus P30 protein monoclonal antibody and preparation method thereof
  • Kit for screening and detecting African swine fever virus P30 protein monoclonal antibody and preparation method thereof

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Experimental program
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Effect test

Embodiment 1

[0026] The preparation of embodiment 1 African swine fever virus P30 protein

[0027] 1 material

[0028] 1.1 Strains and vectors Competent cells of Escherichia coli BL21 were purchased from Bomed Technology Co., Ltd.; gene synthesis was synthesized by General Biosystems (Anhui) Co., Ltd. (providing plasmid PET-30a).

[0029]1.2 The main reagent DNAMarker was purchased from Thermo Company, and the plasmid mini-extraction kit was purchased from AXYGEN Company; other chemical reagents were domestic analytical grade.

[0030] 1.3 Instruments Centrifμge 5804R desktop high-speed centrifuge was purchased from Eppendorf, Germany; PCR instrument, nucleic acid electrophoresis instrument, VerSaDoc2000 gel imaging system were purchased from Bio-Rad, USA; HZQ-C air bath constant temperature double-layer oscillator was purchased from Harbin Dongming Medical Instrument factory; SW-CJ-IF ultra-clean bench, Antai Company of Sujing Group; -80℃ refrigerator, Sanyo, Japan.

[0031] 1.4 Prepara...

Embodiment 2

[0044] Embodiment 2 Establishment of the kit for identification of African swine fever virus P30 protein monoclonal antibody

[0045] 1 Materials and methods

[0046] 1.1 Test material

[0047] 1.1.1 The purified African swine fever virus P30 protein was prepared by Zhejiang Sci-Tech University.

[0048] 1.1.2 ELISA plate was purchased from Corning Company of the United States.

[0049] 1.1.3 Goat anti-mouse enzyme-labeled antibodies were purchased from Huamei Biological Company.

[0050] 1.1.4 The main reagents TMB single-component chromogenic solution was purchased from Beijing Suo Laibao Technology Co., Ltd.; other chemical reagents were domestic analytical grade.

[0051] 1.1.5 Freund's complete adjuvant and Freund's incomplete adjuvant were purchased from Sigma.

[0052] 1.1.6 BALB / c mice were purchased from Yangzhou University.

[0053] 1.2 Test method

[0054] 1.2.1 Preparation and testing of positive serum and negative serum

[0055] 1.2.1.1 Preparation of negat...

Embodiment 3

[0106] The detection method of embodiment 3 African swine fever virus P30 protein monoclonal antibody identification kit

[0107] 1. Sample dilution: use the sample diluent to dilute the sample to be tested 1:100 times;

[0108] 2. Add samples: Add diluted samples, negative control and positive control, among which, positive control and negative control are repeated twice, 100 μl / well, incubated at 37°C for 1 hour, washed 3 times with 1×PBST, 300 μl per well , 3 minutes each time, and pat dry for the last time;

[0109] 3. Add goat anti-mouse enzyme-labeled antibody: add goat anti-mouse enzyme-labeled antibody to each reaction well, 100 μl / well, incubate at 37°C for 1 hour, wash 5 times with 1×PBST, 300 μl per well, 3 minutes each time, and finally Pat dry once;

[0110] 4. Add chromogenic solution: add 100 μl chromogenic solution to each reaction well, and develop color at 37°C in the dark for 10 minutes;

[0111] 5. Add stop solution: add 50 μl stop solution to each react...

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Abstract

The invention discloses an ELISA kit for identifying an African swine fever virus P30 protein monoclonal antibody and a preparation method and a use method of the kit. The kit provided by the invention can be used for large-scale screening and detection of the African swine fever virus P30 protein monoclonal antibody on the premise of not using the African swine fever virus.

Description

technical field [0001] The invention relates to an ELISA kit for identifying the monoclonal antibody of African swine fever virus P30 protein, a detection method and a preparation method thereof. It belongs to the field of biomedicine, specifically to the field of virus disease diagnosis technology and animal quarantine. Background technique [0002] African swine fever (English name: African Swine fever, referred to as ASF) is caused by African swine fever virus (English name: African Swine fevervirus, referred to as ASFV) infecting domestic pigs and various wild boars (such as African wild boars, European wild boars, etc.) An acute, hemorrhagic, severe infectious disease. The World Organization for Animal Health (OIE) listed it as a legally notifiable animal disease, and this disease is also a class of animal epidemics that my country focuses on preventing. It is characterized by a short course of disease, the most acute and acute infection mortality rate is as high as 1...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N33/58G01N33/569G01N33/543G01N33/531
CPCG01N33/6854G01N33/581G01N33/56983G01N33/54306G01N33/531G01N2333/01G01N2469/20
Inventor 孙统威周晶晶徐慧珍
Owner 杭州恒奥科技有限公司
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