African swine fever virus magnetic particle chemiluminiscence antibody detection kit and application thereof

An African swine fever virus and chemiluminescence technology, which is applied in the field of African swine fever virus magnetic particle chemiluminescence antibody detection kit, can solve the problems of low sensitivity, reducing the area of ​​antigen or antibody interaction, etc.

Inactive Publication Date: 2020-10-20
LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] In order to overcome the low sensitivity of the above-mentioned detection technology and the problem that the area of ​​action of antigen or antibod...

Method used

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  • African swine fever virus magnetic particle chemiluminiscence antibody detection kit and application thereof
  • African swine fever virus magnetic particle chemiluminiscence antibody detection kit and application thereof
  • African swine fever virus magnetic particle chemiluminiscence antibody detection kit and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Embodiment 1 The formation of African swine fever virus magnetic particle chemiluminescent antibody detection kit

[0026] 1. Experimental method

[0027] 1.1 Preparation of ASFV p30 recombinant protein

[0028] 1.1.1 Propagation of strains for P30 recombinant protein production

[0029] Take the frozen preserved original strain E.coli-ASFV-p30 and inoculate it in LB liquid medium containing kanamycin (100 μg / mL), culture it at 37°C until the OD600nm value is 0.65, add 20% of the total volume after sampling Store in sterile glycerol aliquots.

[0030] 1.1.2 Prokaryotic expression of p30 recombinant protein

[0031] Preparation of bacterial solution: use 1 basic seed of genetically engineered bacteria E.coli-ASFV-p30, take an appropriate amount from the inoculation loop and streak on LB agar plate (containing 100 μg / mL kanamycin), culture at 37°C for 12 hours, as a primary seed. The primary seeds (single colony) were inoculated in 3 mL of LB medium (containing 100 μ...

Embodiment 2

[0108] Example 2 Detection and determination of African swine fever virus magnetic particle chemiluminescent antibody detection kit

[0109] 1. Test method

[0110] 1.1 Optimal reaction time selection

[0111] The reaction time of each step is 3 gradients of 5min, 10min and 15min. Detection by automatic chemiluminescence immunoassay analyzer. Determine the optimal reaction time based on the luminescence value.

[0112] 1.2 Determination of optimal sample volume

[0113] 1.2.1 Determination of the optimal sample volume of serum

[0114] For the detection of standard serum, the serum sample volume was set at 10 μL and 20 μL respectively, and detected with a fully automatic chemiluminescence immunoassay analyzer. According to the luminescence value and the detection range, determine the optimal amount of serum to add.

[0115] 1.2.2 Determination of the optimal amount of sample diluent

[0116] For the detection of standard serum, the amount of sample diluent was set to 10...

Embodiment 3

[0161] Example 3 Performance index of African swine fever virus magnetic particle chemiluminescent antibody detection kit

[0162] 1. Test method

[0163] 1.1 Specificity experiment

[0164] With 3 batches of ASFV antibody magnetic particle chemiluminescence kits prepared, to ASFV antibody positive serum (20 parts); ASFV antibody negative serum (10 parts); Seneca virus positive serum 5 parts; Classical swine fever virus positive serum 5 parts; 5 positive sera of reproductive and respiratory syndrome virus; 5 positive sera of foot-and-mouth disease virus type O; .

[0165] 1.2 Sensitivity test

[0166] The positive standard serum was diluted with the sample diluent at a ratio of 1:2, 1:8, and 1:32, respectively, and named CP1, CP2, and CP3, respectively. As sensitive quality control serum, three batches of ASFV Ab magnetic particle chemistry were used. Luminescence kit detection. The S / CO of CP1 is required to be greater than 5.0, the S / CO of CP2 is between 1 and 2, and the ...

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Abstract

The invention belongs to the technical field of immunoassay analysis, and particularly relates to an African swine fever virus magnetic particle chemiluminescence antibody detection kit and application thereof. The kit uses ASFV P30 recombinant protein as a magnetic particle conjugate, and uses goat anti-pig IgG marked by a chemiluminescence marker as a detection antibody. The kit is specificallycomposed of a magnetic particle suspension coupled with ASFV P30 recombinant protein, a reagent R, a calibration product, a quality control product, a sample diluent, a washing solution and a luminescent solution, wherein the reagent R is a diluted chemiluminescent marker labeled goat anti-pig IgG antibody working solution. The kit disclosed by the invention takes a magnetic particle chemiluminescence method as a detection technology, is combined with an alkaline phosphatase labeling technology, has the characteristics of high specificity, good sensitivity, excellent repeatability, strong stability and the like, and can be widely applied to basic ASFV detection.

Description

technical field [0001] The invention belongs to the technical field of immunoassay detection and analysis, and in particular relates to an African swine fever virus magnetic particle chemiluminescent antibody detection kit and an application thereof. Background technique [0002] African swine fever virus (African swine fever virus, ASFV) belongs to single-stranded double-stranded DNA virus, and its infection pig can cause high fever, skin cyanosis and severe hemorrhage of lymph nodes and internal organs of pigs, and the acute type mortality rate can reach 100%, and African swine fever does not always show full clinical symptoms, and its symptoms are easily confused with those of other diseases in the early stages of the disease or when a small number of animals are infected. After the outbreak of the disease in my country in August 2018, it not only seriously troubled the prevention and control of animal diseases in my country, but also seriously jeopardized the development...

Claims

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Application Information

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IPC IPC(8): G01N33/96G01N33/569G01N33/543G01N33/535
CPCG01N33/535G01N33/54326G01N33/56983G01N33/96G01N2333/01G01N2469/20
Inventor 郑海学田宏罗俊聪石正旺王丽娟万颖宋锐杨波张克山李丹茹毅杨帆朱紫祥曹伟军党文
Owner LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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