Newcastle disease virus double-antibody sandwich AlphaLISA detection kit and detection method thereof

A newcastle disease virus and double-antibody sandwich technology, which is applied in the field of agricultural biology, can solve problems such as unsuitable for promotion, and achieve good titer, good stability, and good detection reliability.

Inactive Publication Date: 2016-07-13
CHONGQING UNIV OF TECH
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  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

CN1827775A (patent application number is 200510008997.6) "Nucleoside sequence, kit and test method for detection of Newcastle virus", the invention selects Newcastle disease virus F gene (synthetic F protein) as the target region, and the selection can reflect the characteristics of the cleavage site and lack Design multiple pairs of primers and probes for the conserved region of the secondary structure, and establish fluorescent PCR to detect Newcastle disease virus. This method is 100-1000 times more sensitive than the traditional PCR method, but it takes about 4 hours from sample processing to the result. This method The implementation also needs expensive instruments such as fluorescence quantitative PCR instrument, so it is not suitable to be promoted at the grassroots level

Method used

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  • Newcastle disease virus double-antibody sandwich AlphaLISA detection kit and detection method thereof
  • Newcastle disease virus double-antibody sandwich AlphaLISA detection kit and detection method thereof
  • Newcastle disease virus double-antibody sandwich AlphaLISA detection kit and detection method thereof

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Experimental program
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Effect test

Embodiment 1

[0017] The purification of embodiment 1 Newcastle disease virus antigen

[0018] Dilute Newcastle disease virus AV29 standard strain (NDV-AV29) (purchased from China Veterinary Drug Administration) with sterilized PBS buffer (pH7.4, 0.01M) at a volume ratio of 1:50, and inoculate the allantoic cavity 9-10 day-old SPF chicken embryos, the allantoic fluid of dead chicken embryos was harvested after 24 hours. A total of 1250 mL of NDV allantoic fluid was collected, and 1% (volume concentration) chicken red blood cells were prepared, and the hemagglutination titer of the collected virus allantoic fluid was measured to reach 1:128-1:256.

[0019] The harvested virus allantoic fluid was purified successively by differential centrifugation and gel filtration, the concentration of virus protein was determined by BCA method, the purity was identified by SDS-PAGE electrophoresis, and the hemagglutination titer of the virus was determined by hemagglutination test.

[0020] 1. Purificati...

Embodiment 2

[0028] Example 2 Preparation of Anti-Newcastle Disease Virus Hemagglutinin Protein Monoclonal Antibody

[0029] 1. Establishment of mouse anti-Newcastle disease virus hemagglutinin protein monoclonal antibody hybridoma cell line

[0030] The above-mentioned purified NDV was immunized to BALB / c mice, and splenocytes after 4 times of immunization were fused with SP2 / 0 myeloma cells with PEG-1500. The first fusion rate was 86.5%, and the second fusion rate was 86.5%. 90.1%. To establish indirect ELISA, the purified NDV and blank allantoic fluid are coated with ELISA plate at the same time for ELISA screening. The positive wells screened by the blank allantoic fluid are false positive wells, which should be excluded. Cloned antibody, the initial positive rate was 12.3% and 17.9%. From the positive wells screened by the purified NDV antigen, 42 wells with higher positive values ​​were selected for subcloning. After 2 subclonings and repeated screening, 5 hybridoma cell lines with...

Embodiment 3

[0046] Example 3 Newcastle disease virus double antibody sandwich AlphaLISA detection kit and its detection method

[0047] 1. Preparation of reference standard

[0048] Dilute the NDV antigen to a concentration of 0, 0.1ng / mL, 1ng / mL, 10ng / mL, 100ng / mL, 1000ng / mL, the dilution solution contains 50mmol / LTris-HCl, mass concentration 1.5% BSA, mass concentration 0.9% NaCl, mass concentration 0.05% NaN3, mass concentration 0.01% Tween-20, pH 7.8 reaction buffer.

[0049] 2. Establishment of AlphaELISA system

[0050] The prepared mouse anti-NDV hemagglutinin monoclonal antibody 2A5 was coated with acceptor microspheres, and the acceptor microspheres connected with antibody 2A5 were mixed with 1xAphaLISA buffer (10xAlphaLISAassayBuffer was diluted into 1xAlphaLISAassayBuffer with ultrapure water, and the following The same) was diluted at a volume ratio of 1:200, and the prepared mouse anti-NDV hemagglutinin protein monoclonal antibody 4E11 was used for biotin labeling, and the ...

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Abstract

The invention provides a Newcastle disease virus double-antibody sandwich AlphaLISA detection kit and a detection method thereof. The detection kit comprises a receptor microballoon coated with a Newcastle disease virus antibody, a biotin labeled Newcastle disease virus antibody and a donor microballoon coated with streptavidin. The detection method comprises the following steps of: successively adding a standard substance or a to-be-detected sample, the receptor microballoon coated with the Newcastle disease virus antibody and the biotin labeled Newcastle disease virus antibody into a porous plate and performing vibration incubation; adding the donor microballoon coated with streptavidin under a light shielding condition and then performing vibration incubation; and detecting a signal value on a AlphaScreen/Lisa detector. The detection kit provided by the invention has high stability and strong specificity. The influence of outside environment on the detection method is small. The detection method has higher sensitivity and specificity, can meet the requirements of high throughput and quick detection and has high popularization and application value.

Description

technical field [0001] The invention belongs to the field of agricultural biotechnology, relates to veterinary medicine, immunology and biological products, in particular to a Newcastle disease virus double-antibody sandwich AlphaLISA detection kit and a detection method thereof. Background technique [0002] Newcastle disease (Newcastle disease, ND) is a serious disease that harms poultry and is listed as a class A infectious disease by the International Veterinary Bureau. In the "National Medium and Long-Term Animal Disease Prevention and Control Plan (2012-2020)", Newcastle disease is listed as one of the major animal diseases that are prioritized for prevention and control. Some clinical symptoms of the disease are similar to those of avian influenza, sometimes resulting in immeasurable economic losses due to misdiagnosis. Therefore, rapid and accurate detection of the disease is particularly important. [0003] At present, the diagnosis or detection of Newcastle diseas...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/577G01N33/569
CPCG01N33/577G01N33/56983G01N2333/125
Inventor 吴胜昔王玲玲蔡家利罗彬彬龙京慧李袁元张中豪周康森顾盼
Owner CHONGQING UNIV OF TECH
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