Method for preparing fluorescent antibody for detecting avian influenza virus and solid phase immunofluorescence detection kit

An avian influenza virus and fluorescent antibody technology, applied in the field of immunofluorescence detection, can solve the problems of reduced fluorescence detection sensitivity, low background fluorescence, low sensitivity, etc., and achieve the effects of good antibody activity, bright fluorescence, and high cross-linking efficiency.

Inactive Publication Date: 2011-01-26
INST OF ANIMAL SCI & VETERINARY MEDICINE SHANDONG ACADEMY OF AGRI SCI
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Problems solved by technology

The above methods have their own advantages and disadvantages: some are time-consuming, such as virus isolation and serological identification; High technical requirements, such as fluorescent PCR; some simple and easy, but low sensitivity, such as HA-HI
Carriers commonly used in solid-phase immunofluorescence detection include nylon membranes, nitrocellulose membranes, cellulose acetate membranes, 96-well plates, etc., but commonly used solid-phase carriers have low background fluorescence, which will reduce the sensitivity of fluorescence detection

Method used

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  • Method for preparing fluorescent antibody for detecting avian influenza virus and solid phase immunofluorescence detection kit
  • Method for preparing fluorescent antibody for detecting avian influenza virus and solid phase immunofluorescence detection kit
  • Method for preparing fluorescent antibody for detecting avian influenza virus and solid phase immunofluorescence detection kit

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Embodiment approach 1

[0029] Embodiment 1: A method for detecting H 9 Preparation method of fluorescent antibody of subtype avian influenza virus and solid-phase immunofluorescence detection kit thereof

[0030] Preparation of allophycocyanin (APC): add 5 times the volume (v / w) of 20mM phosphate buffer (pH6.8-7.0) to Spirulina platensis cells, freeze and thaw 3 times, centrifuge at 10000rpm at 4°C, Ammonium sulfate was added to the supernatant to a final concentration of 60% (w / v), placed in a refrigerator at 4°C for 24 hours, centrifuged, and the precipitate was dissolved in 20 mM phosphate buffer (pH 7), dialyzed in 20 mM phosphate buffer (pH 7), Chromatography on DEAE Sepharose Fast Flow anion-exchange column on the dialyzate, the ion-exchange column was pre-equilibrated with 20mM acetate buffer (pH5.0, containing 50mM NaCl), and the eluent was 20mM acetate buffer (pH3.6, containing 50mM NaCl) , the elution rate is 60mL / h, the collected sky blue liquid is the purified spirulina allophycocyanin,...

Embodiment approach 2

[0045] Embodiment 2: Detection of H 5 Preparation method of fluorescent antibody of subtype avian influenza virus and solid-phase immunofluorescence detection kit thereof

[0046] Preparation of allophycocyanin (APC): Collect algae cells of Spirulina platensis, add 5 times the volume (v / w) of 20mM phosphate buffer (pH6.8-7.0), freeze and thaw three times , centrifuged at 10000rpm at 4°C, added ammonium sulfate to the supernatant to a final concentration of 60% (w / v), placed in a refrigerator at 4°C for 24 hours, centrifuged, and the precipitate was dissolved in 20mM phosphate buffer (pH7), and 20mM phosphate buffer The dialysate was dialyzed against DEAE Sepharose Fast Flow anion-exchange chromatography column, the ion-exchange column was pre-equilibrated with 20mM acetate buffer (pH5.0, containing 50mMNaCl), and the eluent was 20mM acetate buffer (pH3. 6, containing 50mM NaCl), the elution rate is 60mL / h, the collected sky blue liquid is the purified spirulina allophycocyani...

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Abstract

The invention relates to a method for preparing a fluorescent antibody for detecting avian influenza virus and a solid phase immunofluorescence detection kit. According to the invention, an avian influenza virus antibody is marked by allophycocyanin (APC) so as to prepare the avian influenza virus fluorescent antibody; the APC and the antibody are derived by a chemical cross-linking agent SPDP respectively; the derivatives are subjected to liquid phase cross-linking in a proper molar ratio; and then the fluorescent antibody is prepared by high pressure liquid chromatography purification. The solid phase immunofluorescence detection kit comprises the fluorescent antibody, CNBr activated agarose microspheres, the avian influenza virus antibody, cleaning solution and the like. The using method of the kit comprises the following steps of: activating a microsphere carrier and then coating the activated microsphere carrier by using the avian influenza virus antibody; cleaning; combining the microspheres coated with the antibody with a sample (an antigen) to be detected; combining the microspheres coated with the antibody with the fluorescent antibody after cleaning; removing the uncombined fluorescent antibody by cleaning; and observing under a fluorescence microscope and determining the result. The fluorescent antibody prepared by the method is characterized by high cross-linking efficiency, high purity, bright red fluorescence and stable performance. The microsphere solid phase carrier which is adopted by the kit can obviously improve fluorescent detection sensitivity, and is suitable for the quick detection of the avian influenza virus.

Description

technical field [0001] The invention relates to a preparation method of a fluorescent antibody for detecting avian influenza virus and a solid-phase immunofluorescence detection kit, belonging to the field of immunofluorescence detection. technical background [0002] Avian influenza is a severe infectious disease caused by avian influenza virus that seriously harms poultry, livestock and wild animals, and even threatens human health. Rapid and sensitive detection methods are crucial for timely diagnosis of the disease, effective prevention and control of the epidemic, and prediction and early warning. The detection methods of avian influenza mainly include: (1) virus isolation and identification; (2) serological detection, such as HA-HI, ELISA, etc.; (3) molecular biological detection, such as RT-PCR. The above methods have their own advantages and disadvantages: some are time-consuming, such as virus isolation and serological identification; High technical requirements, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/569G01N33/533G01N21/64C07K16/10C07K16/06
Inventor 颜世敢朱丽萍李雁冰张玉忠周百成胡北侠张秀美许传田杨少华
Owner INST OF ANIMAL SCI & VETERINARY MEDICINE SHANDONG ACADEMY OF AGRI SCI
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