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Chicken infectious bronchitis immune body immune colloidal gold fast detecting reagent kit and its application

A technology for bronchitis and chicken infectivity, applied in the field of immunization, can solve the problems of impracticability, undisclosed biological material samples, and failure to submit biological material samples for preservation, etc., and achieve the effect of high biological safety and low production cost

Inactive Publication Date: 2008-06-11
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The utility model patent application for chicken infectious bronchitis virus antibody detection kit (application number 02285790.7) is an ELISA method, and does not disclose the biological material samples used in it, nor does it submit the preservation of biological material samples for patent procedures. The skilled person is unable to implement the invention and achieve the effect indicated by the invention

Method used

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  • Chicken infectious bronchitis immune body immune colloidal gold fast detecting reagent kit and its application
  • Chicken infectious bronchitis immune body immune colloidal gold fast detecting reagent kit and its application
  • Chicken infectious bronchitis immune body immune colloidal gold fast detecting reagent kit and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Preparation of Chicken Infectious Bronchitis NP Protein

[0028] For the molecular biology methods used, please refer to the literature: Sambrook J, Fritsch EF, Mani Artis T, editors (translated by Jin Dongyan, etc.). Molecular Cloning Experiment Guide. The second edition, provided by Beijing Science Press, 1992 method is carried out.

[0029] 1. Cloning and sequencing of NP gene

[0030] The NP gene involved in the present invention is obtained by cloning the chicken infectious bronchitis H52 strain in the Microbiology and Immunity Laboratory where the applicant works. The sequence of the gene has been registered in the GeneBank database with the accession number DQ473615. The sequence has 97%-99% homology with known sequences. The specific method of cloning and sequencing of NP gene is: use chicken infectious bronchitis H52 strain (Li Zhonghua, Infectious bronchitis virus spike protein, nucleoprotein gene cloning, expression and its preliminary application in antibo...

Embodiment 2

[0036] Preparation of rabbit anti-mouse IgG antibody:

[0037] 1. Extraction of Balb / C mouse IgG

[0038] The extraction and purification of IgG are carried out according to the methods described in Shen Guan et al. (Shen Guan et al., Modern Immunology Experimental Technology (Second Edition) [M], Hubei Science and Technology Press, 2002): Take 10.0mL healthy Balb / C small After the mouse serum was mixed with an equal volume of normal saline, saturated (NH 4 ) 2 SO 4 Solution 20.0mL, made to 50% saturation (NH 4 ) 2 SO 4 solution. Take it out after standing at 4°C for 30min; centrifuge at 3,000r / min for 30min at 4°C, discard the supernatant, add 20.0mL normal saline to the precipitate; after the precipitate dissolves, slowly add 10.0mL saturated (NH 4 ) 2 SO 4 solution, made into 33% saturation (NH 4 ) 2 SO 4 solution. Take it out after standing at 4°C for 30min; centrifuge at 3,000r / min for 30min at 4°C. Discard the supernatant and precipitate. Repeat the above o...

Embodiment 3

[0053] Preparation of nitrocellulose membrane

[0054] 1. Preparation of coating buffer: 0.01M pH7.2 PBS buffer containing 3% methanol is used as coating buffer, filtered through 0.22μ membrane, set at 4°C for later use, valid for one week. 1000ml 3% methanol 0.01M pH7.2 PBS buffer formulation: NaCl 8g, KCl 0.2g, NaCl 2 HPO 4 12H2O 2.9g, KH 2 PO 4 0.2g, methanol 30ml, distilled deionized water to 1000ml.

[0055] 2. Preparation of nitrocellulose membrane: use the coating buffer in step 1 of this example to dilute the NP protein to 50-100 μg / ml, adjust the machine, draw the T line, which is the detection line, and the T line is close to the gold The end of the standard pad is about 5mm away from the end of the gold standard pad; dilute the rabbit anti-mouse IgG antibody to 50-100 μg / ml with coating buffer, adjust the machine, and draw the line C, which is the quality control line, and the C line is close to the absorption pad, about 3mm from the absorbent pad. The distan...

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Abstract

The invention belongs to the immunity application field, which relates to the related fields of animal molecular biochemistry and the immunology, etc. The invention discloses a reagent kit respectively used for antibody of infectious bronchitis of chicken, which comprises a case body, and the test paper card and sample diluent equipped inside the case body, wherein, the test paper card is formed by the absorption pad, nitrocellulose membrane, gold-label pad and sample pad affixed in turn on the non-absorbent supporting flake by taking the sample pad, absorption pad and the NP recombinant proteins coated with the nucleocapsid of the infectious bronchitis of chicken as the detection line and the NP recombinant proteins coated with anti-mouse IgG as nitrocellulose membrane of the quality control line. The reagent kit used for testing the corresponding antibody has the obvious advantages of strong specificity, high sensitivity, easy operation and fast diagnosis.

Description

technical field [0001] The invention belongs to the field of immune application and relates to related fields such as animal molecular biology and immunology. Specifically, the invention belongs to a kit for rapid detection of chicken infectious bronchitis virus antibody, mainly to determine whether the tested chicken has been infected with chicken infectious bronchitis virus or whether the corresponding antibody has been produced in the immunized chicken. Background technique [0002] As we all know, avian influenza, Newcastle disease, bursa, infectious bronchitis, infectious laryngotracheitis, mycoplasma gallisepticum, Marek's disease, egg drop syndrome and other diseases are important diseases that endanger the global poultry industry. [0003] Chicken infectious bronchitis (Infectious bronchitis, IB) is an acute, highly contagious infectious disease of chickens caused by (infectious bronchitis virus, IBV), which can cause various diseases such as the respiratory tract, f...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/577G01N33/569G01N33/558
Inventor 毕丁仁肖运才张进良胡思顺张文通张展英王政李自力王喜亮石德时刘梅许青荣
Owner HUAZHONG AGRI UNIV
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