Double-antibody sandwich ELISA based on African swine fever virus p72 gene and application of double-antibody sandwich ELISA

An African swine fever virus and antibody technology, applied in the field of biotechnology detection, to achieve the effects of high titer, high antigen purity, good immunogenicity and antigenicity

Active Publication Date: 2021-07-23
CHINESE ACAD OF INSPECTION & QUARANTINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although there is no ASFV vaccine at present, a positive ASFV antibody indicates current or past infection, but it does not mean that there will be no ASFV vaccine in the future. Therefore, the detection of ASFV antigen is particularly important. Only by accurately determining the infection situation can we take timely measures to prevent and control the disease measure

Method used

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  • Double-antibody sandwich ELISA based on African swine fever virus p72 gene and application of double-antibody sandwich ELISA
  • Double-antibody sandwich ELISA based on African swine fever virus p72 gene and application of double-antibody sandwich ELISA
  • Double-antibody sandwich ELISA based on African swine fever virus p72 gene and application of double-antibody sandwich ELISA

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Preparation and identification of embodiment 1 African swine fever virus p72 recombinant protein

[0060] 1. Materials

[0061] 1.1 Strains

[0062] Escherichia coli competent DH5α and DH10Bac were purchased from Beijing Quanshijin Biotechnology Co., Ltd., pCMV vector, pFastbac1 vector, and 293F cells were preserved by the Animal Inspection and Quarantine Laboratory of the Chinese Academy of Inspection and Quarantine.

[0063] 1.2 Serum

[0064] The African swine fever standard positive serum inactivated by 0.3% TNBP and 1% triton X-100 was preserved by the Polish National Veterinary Institute. Porcine pseudorabies live vaccine (Kartha-K61 strain) (Pseudorabies virus, PRV) was purchased from Qingdao Yibang Bioengineering Co., Ltd.; porcine parvovirus inactivated vaccine (WH-1) (porcine parvovirus, PPV) was purchased from Wuhan Keqian Biological Co., Ltd.; porcine circovirus type 2 inactivated vaccine (LG strain) (porcine circovirus, PCV2) was purchased from Harbin Ve...

Embodiment 2

[0109] Embodiment 2 Preparation of anti-African swine fever virus p72 protein mouse monoclonal antibody

[0110] 1. Materials

[0111] 1.1 virus: porcine pseudorabies live vaccine (Kartha-K61 strain) (Pseudorabies virus, PRV), purchased from Qingdao Yibang Bioengineering Co., Ltd.; Porcine parvovirus inactivated vaccine (WH-1) (porcine parvovirus, PPV), Purchased from Wuhan Keqian Biotechnology Co., Ltd.; porcine circovirus type 2 inactivated vaccine (LG strain) (porcine circovirus, PCV2) was purchased from Harbin Veken Biotechnology Development Company, live swine fever vaccine (C strain) (Classical Swine Fever, CSF), highly pathogenic porcine reproductive and respiratory syndrome live vaccine (HuN4-F122 strain) (Porcine reproductive and respiratory syndrome, PRRS) were purchased from Harbin Pharmaceutical Group Biological Vaccine Co., Ltd. The Institute of Animal Inspection and Quarantine of the Chinese Academy of Sciences isolated, identified and preserved the vaccine.

...

Embodiment 3

[0163] Example 3 Purification, Identification and Matching Test of Monoclonal Antibodies

[0164] 1. Preparation of ascites from hybridoma cell lines

[0165] Take 8-10 week-old multiparous BALB / c mice and inject 0.5 mL of special adjuvant for ascites intraperitoneally. After 12-15 days, expand the screened hybridoma cell lines, collect hybridoma cells, and centrifuge at 1000rpm for 10min. Resuspend with an appropriate amount of sterile PBS, and intraperitoneally inject 0.2mL hybridoma cells / mouse (containing about 2.5×10 61 cell), about 10-15 days later, when the abdominal circumference of the mouse increased, ascites was collected with a sterile syringe needle. Centrifuge the collected ascites at 10,000r / min for 10min, collect the middle layer, and filter it with a 0.45μm filter to obtain the corresponding monoclonal antibody (ascites). Take the supernatant and press: 1:1000, 1:2000, 1:4000 , 1:8000, 1:16000, 1:32000, 1:64000, 1:128000, 1:256000, 1:512000 gradient dilution...

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Abstract

The invention discloses an antigen conjugated protein specifically conjugated with an African swine fever virus p72 protein, an antibody or active fragment and a double-antibody sandwich ELISA antigen detection kit for the African swine fever virus p72 protein containing the antigen conjugated protein, the antibody or the active fragment and application of the double-antibody sandwich ELISA antigen detection kit. The kit disclosed by the invention is coated with an ELISA plate, positive control and negative control of a monoclonal antibody ASFV-p72-5G8, an HRP-labeled detection antibody ASFV-p72-12F6, a sample diluent, color developing liquid and washing liquid. In which, the antibody ASFV-p72-5G8 and the antibody ASFV-p72-12F6 are secreted from hybridoma cell strains ASFV-p72-5G8 and ASFV-p72-12F6 separately. According to the kit disclosed by the invention, a double-antibody sandwich ELISA method is established by taking 5G8 as a capturing antibody and taking 12F6-coupled HRP as a detection antibody, can be used for specifically detecting ASFV, is simple and convenient in operation, high in sensitivity and good in specificity and provides a novel detecting means for clinical diagnosis and epidemiological survey on ASFV infection.

Description

technical field [0001] The invention belongs to the field of biotechnology detection, and in particular relates to a double-antibody sandwich ELISA antigen detection kit based on the p72 gene of African swine fever virus and its application in detection of fresh blood of sick animals. Background technique [0002] African swine fever (African swine fever, ASFV) is an acute, virulent, highly contagious disease of pigs caused by African swine fever virus (ASFV). swine fever (CSF) is highly similar and originated in Africa, so it is called African swine fever. [0003] In recent years, with the development of monoclonal antibody technology, the research on African swine fever monoclonal antibody has played an increasingly prominent role in ASFV research and ASFV diagnosis, mainly in two aspects. One is to combine monoclonal antibody For the establishment of immunology-related ASFV detection methods, such as direct immunofluorescence test (DIF), immunohistochemistry test, sandw...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/08G01N33/569G01N33/577G01N33/58G01N33/543
CPCC07K16/081G01N33/56983G01N33/577G01N33/581G01N33/54393C07K2317/565C07K2317/56C07K2317/92C07K2317/33G01N2333/01G01N2469/10
Inventor 冯春燕林祥梅王彩霞吴绍强刘晓飞仇松寅
Owner CHINESE ACAD OF INSPECTION & QUARANTINE
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