Newcastle disease immune body immune colloidal gold fast detecting reagent kit and its application

A chicken Newcastle disease and kit technology, applied in the field of immunization applications, can solve the problems of easy to disperse, poor biological safety, and inability to implement, and achieve the effects of high biological safety and low production cost.

Active 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

Patent application for manufacturing method of gold standard test strips for semi-quantitative and rapid detection of Newcastle disease antibody (application number 200610042408.0), which uses colloidal gold-labeled Newcastle disease virus as the gold standard pad and Newcastle disease virus as the detection line. Some disadvantages of this method are: purification of the virus It is not easy to standardize, and it is easy to disperse the poison, and the biological safety is poor; its specificity and sensitivity are not easy to control, and the differences between batches and within batches are also large. Without the deposit of a sample of biological material submitted for patent procedure, a person skilled in the art would not be able to practice the invention and achieve the effect indicated by the invention

Method used

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  • Newcastle disease immune body immune colloidal gold fast detecting reagent kit and its application
  • Newcastle disease immune body immune colloidal gold fast detecting reagent kit and its application
  • Newcastle disease immune body immune colloidal gold fast detecting reagent kit and its application

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Experimental program
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Effect test

Embodiment 1

[0026] Preparation of HN Protein of Chicken Newcastle Disease Virus

[0027] For the molecular biology methods used, please refer to the literature: Sambrook J, Fritsch EF, Mani Artis T, editors (translated by Jin Dongyan, Li Mengfeng, etc.), Molecular Cloning Experiment Guide, Second Edition, Beijing Science Press, 1992 method provided in.

[0028] 1. Cloning and sequencing of HN gene

[0029] The HN gene involved in the present invention is obtained by the applicant's own clone according to the HN gene sequence of NDV La Sota line included in GenBank, and the sequence has 98% homology with the known sequence. The specific method of cloning and sequencing of the HN gene is: use chicken Newcastle disease (NDV) Losota strain, purchase from the Lanzhou Veterinary Research Institute of the Chinese Academy of Agricultural Sciences, or refer to references: Sun Shuna, Newcastle disease virus F gene and HN gene clone expression and its Preliminary establishment of ELISA antibody de...

Embodiment 2

[0038] Preparation of rabbit anti-mouse IgG antibody:

[0039] 1. Extraction of Balb / C mouse IgG

[0040] 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 mouse serum was mixed with an equal amount of 0.8% sodium chloride solution, 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 of 0.8% sodium chloride solution to the precipitate; after the precipitate dissolves, slowly add 10.0mL of 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, ...

Embodiment 3

[0054] Preparation of nitrocellulose membrane

[0055] 1 Preparation of coating buffer: 0.01M pH7.2 PB buffer containing 3% methanol was used as coating buffer, filtered through 0.22μ membrane, set at 4°C for later use, and valid for one week. 1000ml 3% methanol 0.01M pH7.2 PB buffer formulation: KCl 0.2g, Na 2 HPO 4 12H 2 O 2.9g, KH 2 PO 4 0.2g, methanol 30ml, distilled deionized water to 1000ml.

[0056] 2 Preparation of nitrocellulose membrane: Dilute the HN protein to 50-100 μg / ml with coating buffer, adjust the machine, and mark the line as T line, that is, the detection line (T line) is close to the end of the gold standard pad, and the distance from the gold standard The end of the pad is about 5mm; dilute the rabbit anti-mouse IgG antibody to 50-100μg / ml with the coating buffer, adjust the machine, draw the line C, which is the quality control line, and the C line is close to the absorbent pad, about 3mm away from the absorbent pad . The distance between the tw...

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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 used for fast testing the antibody of Newcastle Disease, 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 HN recombinant proteins coated with the Newcastle Disease as the detection line and the HN 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 present invention belongs to a rapid detection kit for antibodies against chicken infectious diseases, which is used for the detection of Newcastle disease antibodies in chicken serum, so as to determine whether the tested chickens have been infected with Newcastle disease virus or whether the corresponding antibodies have been produced in the immunized chickens. . Background technique [0002] Newcastle disease (ND), also known as chicken plague, Asian chicken plague, etc., is one of the main poultry infectious diseases that endanger my country's poultry industry. It mainly affects chickens, turkeys, other poultry and wild birds, causing respiratory infections and immunity. Inhibition has been widely existing in various poultry flocks for many years. Although there are various vaccines for immunizati...

Claims

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Application Information

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