African swine fever virus antibody detection test paper established based on capsid protein p72 and preparation method thereof

An African swine fever virus and capsid protein technology, which is applied in the field of immune detection to achieve the effects of strong detection specificity, great promotion and application value, and accurate detection

Pending Publication Date: 2021-11-19
HENAN ACAD OF AGRI SCI
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  • Abstract
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  • Claims
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AI Technical Summary

Problems solved by technology

[0005] At present, there are no vaccines and therapeutic drugs in the prevention and control of ASF, so early detection of infected animals is extremely important to control the outbreak and spread of the disease

Method used

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  • African swine fever virus antibody detection test paper established based on capsid protein p72 and preparation method thereof
  • African swine fever virus antibody detection test paper established based on capsid protein p72 and preparation method thereof
  • African swine fever virus antibody detection test paper established based on capsid protein p72 and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1 Preparation of ASFV-p72 recombinant protein

[0052] According to GenBank: MK333180.1, the gene sequence B646L encoding p72 protein and its partner protein gene sequence B602L were obtained, His and Flag tags were added to the N segment of the protein respectively, and the pCMV vector was constructed, codon optimized, and handed over to Shanghai Sangon Bioengineering Co., Ltd. The company synthesized and obtained two plasmids, named pCMV-B646L and pCMV-B602L respectively.

[0053] The two plasmids were co-transfected into HEK293F cells (at a density of 2.0 × 10 6 cells / mL), after 72 hours of culture, centrifuge at 100g / min for 10 minutes to collect the cells. After discarding the culture medium, add 400mM NaCl and 20mM HEPESBuffer to resuspend the cell pellet, and perform ultrasonic disruption. Then centrifuge at 12000rpm / min for 15min. After centrifugation, the supernatant was filtered through a 0.45um filter membrane, and was washed with 100mM imidazole an...

Embodiment 2

[0057] The preparation of embodiment 2 anti-ASFV-p72 recombinant protein polyclonal antibody

[0058] The purified ASFV-p72 recombinant protein was used as the immunogen to immunize about 2.0kg of healthy New Zealand rabbits. For the first immunization, the antigen was emulsified with complete Freund’s adjuvant, and the dose of 200 μg / rat was injected subcutaneously at multiple points; The antigen was emulsified in Freund's incomplete adjuvant for injection. Two weeks after the last booster immunization, the ASFV-p72 recombinant protein was coated with an enzyme-linked immunosorbent plate at a concentration of 1.6 μg / ml and 50 μL per well, and the antibody titer of the immune serum was determined by ELISA. The titer reaches 1:6.4×10 4 , collect hyperimmune rabbit whole blood, put it at 37°C for 2h, separate the serum at 4°C, centrifuge at 3000rpm / min for 10min to collect the supernatant, and obtain the rabbit polyclonal antibody against ASFV-p72 recombinant protein.

[0059]...

Embodiment 3

[0061] Embodiment 3 African swine fever virus antibody detects the preparation of colloidal gold immunochromatography test paper

[0062] (1) Preparation of colloidal gold solution

[0063] Take 100mL ultrapure water in a 500mL clean beaker, add 1mL 10g / L chloroauric acid and boil; add freshly prepared 1.6mL 10g / L trisodium citrate solution quickly under heating and stirring, observe the color from colorless to blue Color→dark red→bright red until the color no longer occurs. After the solution is cooled to room temperature, dilute to 100mL with ultrapure water and store at 4°C in the dark.

[0064] (2) Colloidal gold labeling of ASFV-p72 recombinant protein

[0065] Taking 1mL colloidal gold solution as an example, add 2μL 0.2mol / L K 2 CO 3 Adjust the pH of the colloidal gold solution, add 120 μL of purified ASFV-p72 recombinant protein to react at room temperature for 5-10 minutes, add 100 μL of 3% casein solution, block at room temperature for 10 minutes, centrifuge at 12...

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Abstract

The invention designs an African swine fever virus antibody detection colloidal gold immunochromatography test paper established based on capsid protein p72 and a preparation method thereof. The test paper comprises a supporting bottom plate, and a sample pad, a gold-labeled protein pad, a chromatography detection membrane and a water absorption pad are sequentially fixed on the supporting bottom plate; the gold-labeled protein pad is coated by glass fiber cotton and is fixed with African swine fever virus p72 recombinant protein labeled by colloidal gold; the chromatographic detection membrane is a nitrocellulose membrane, a detection line and a quality control line are arranged on the nitrocellulose membrane, staphylococcus protein A is fixed on the detection line, and a monoclonal antibody or a polyclonal antibody of anti-African swine fever virus p72 recombinant protein is fixed on the quality control line. The test paper is specific, sensitive, rapid, simple and convenient, can be used for ASFV serological diagnosis and epidemiological investigation, and is easy to popularize and apply in production practice.

Description

technical field [0001] The invention relates to a colloidal gold immunochromatographic test paper for detecting African swine fever virus antibody based on capsid protein p72 and a preparation method thereof, belonging to the technical field of immunodetection. Background technique [0002] Antibody detection of pathogens is the main means to evaluate whether animals are infected with a certain pathogen. After the virus invades the body, the body will produce corresponding specific antibodies. By detecting the presence and content of specific antibodies in blood samples, the virus infection can be indirectly judged. Compared with the direct detection of pathogens, blood samples for antibody detection are easier to obtain and the quality of the samples is guaranteed, the detection sensitivity is high, and it is not easy to miss detection in disease detection and monitoring. The current method for antibody detection is mainly enzyme-linked immunosorbent assay (ELISA), but the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/558G01N33/569
CPCG01N33/558G01N33/56983
Inventor 乔松林耿瑞杨继飞马红芳李睿孙彦刚王磊孙亚宁张改平
Owner HENAN ACAD OF AGRI SCI
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