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Kit for detecting H3N2 subtype canine influenza virus

A technology of canine influenza virus and H3N2, which is applied in the field of immunology, can solve the problems of unfavorable clinical research and development, the lack of H3N2 canine influenza virus, etc., and achieve the effect of high sensitivity, good repeatability and stability, and strong specificity

Active Publication Date: 2015-10-07
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Therefore, it is necessary to strengthen the research on CI; the Chinese patent application number 201410535918.6 discloses a monoclonal antibody against H3N2 subtype canine influenza virus HA2 protein, which is obtained by hybridoma of canine influenza virus isolate JS / 10. Technology has little research on H3N2 canine influenza virus, which is not conducive to clinical research and development

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  • Kit for detecting H3N2 subtype canine influenza virus
  • Kit for detecting H3N2 subtype canine influenza virus
  • Kit for detecting H3N2 subtype canine influenza virus

Examples

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Embodiment 1

[0032] The preparation of embodiment 1 hybridoma cell line

[0033] 1. Antigen preparation of H3N2 subtype canine influenza virus

[0034] The strain A / canine / Guangdong / 1 / 2006 (H3N2) (abbreviated as CGD1) used in the present invention has the Genbank accession numbers GU433345.1, GU433346.1, GU433347.1, GU433348.1, GU433349.1, GU433350 .1, GU433351.1, GU433352.1, strains and their sequence reference website: http: / / www.ncbi.nlm.nih.gov / nuccore / ? term=A%2Fcanine%2FGuangdong%2F1%2F2006%28H3N2%29.

[0035] 1. Virus expansion culture: Inoculate the allantoic cavity of 10-day-old SPF chicken embryos with the diluted chicken embryo allantoic fluid of CGD1 strain, the inoculation volume is 0.15mL / embryo, and incubate at 37°C. Observe once every 12 hours, discard the dead chicken embryos within 24 hours, and store the dead embryos after 24 hours in a refrigerator at 4°C. After 96 hours, all the chicken embryos were cooled overnight at 4°C, the allantoic fluid of the chicken embryos...

Embodiment 2

[0091] The preparation of embodiment 2 monoclonal antibody

[0092] Female BALB / c mice were intraperitoneally injected with FICA, 0.5 mL / mouse. After 7 days, the hybridoma cells 2C5 in the logarithmic growth phase were suspended and washed with sterilized physiological saline (take the supernatant of the cell culture fluid in the logarithmic growth phase to measure the McAb titer), and the cell density was adjusted to 2×10 6 individual / mL. Each mouse was intraperitoneally injected with 0.5mL cell suspension, and observed whether the abdomen of the mouse was swollen day by day. When the ascites was obviously swollen, the ascites was taken out, placed in an EDTA-treated anticoagulant tube, and centrifuged at 5,000r / min for 10min to remove the cells. precipitation. The collected supernatant was inactivated at 56° C. for 30 minutes, centrifuged at 10,000 r / min for 15 minutes, and then purified. Do a good job of disinfection before and after the extraction of mouse ascites, and ...

Embodiment 3

[0126] Example 3 Establishment of Double Antibody Sandwich ELISA Detection Method

[0127] Preparation of rabbit-derived polyclonal antibody: Take two 3kg New Zealand white rabbits, phacoemulsify with purified H3N2 CIV and the same amount of FCA, and inject subcutaneously in multiple points on the back, 1.5mg / rat. The second immunization and the third immunization were emulsified with FICA and H3N2 CIV on the 14th day and 28th day after the first immunization, respectively, and then injected subcutaneously as the antigen, 2 mg per mouse. After the third immunization, blood was collected from the heart, the serum was separated, and stored at -20°C for future use. The titer of the antiserum was determined by indirect ELISA, and the polyclonal antibody concentration after purification (purification steps were the same as in Example 2) was measured using a micro-spectrophotometer, and the purified samples were stained with Coomassie brilliant blue to detect the purification effect...

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Abstract

The invention relates to the technical field of immunology, and concretely discloses a kit for detecting an H3N2 subtype canine influenza virus. The kit includes a monoclonal antibody generated by a hybridoma cell strain H3N2-CIV-HA-2C5, and the hybridoma cell strain is preserved in China Center for Type Culture Collection on May 13, 2015 with the preservation number of CCTCC NO:C201555. The detection kit has the advantages of high sensitivity, strong specificity, very good repeatability and stability, clinic sample detection, realization of a detection rate being greatly higher than that of routine detection, and practical clinic application significance.

Description

technical field [0001] The invention relates to the technical field of immunology, in particular to a kit for detecting H3N2 subtype canine influenza virus. Background technique [0002] Canine influenza (Canine influenza, CI) is a canine animal contact respiratory infectious disease caused by canine influenza virus (CIV), type A influenza virus (IAV) of Orthomyxoviridae family. The disease first broke out in the United States in 2004, and was subsequently reported in Eurasia and other regions. CI is mainly divided into two subtypes, H3N2 and H3N8. The H3N8 subtype is mainly used in Europe and the United States, and the H3N2 subtype is mainly used in most Asian countries. Over the past ten years, IAV has continuously broken through interspecies constraints and cross-infected humans and animals, which has aroused widespread concern about influenza. [0003] Dogs are one of the animals that have the closest contact with humans and may play an important role in the transmissi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/569G01N33/577
CPCG01N33/56983G01N33/577G01N2333/11
Inventor 李守军李陆涛袁子国孙凌霜贾坤远立国卢刚涂黎晴
Owner SOUTH CHINA AGRI UNIV
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