Avian metapneumovirus antibody double-antigen sandwich method ELISA detection kit and application thereof

A double-antigen sandwich and avian metapneumovirus technology, which is applied in the field of avian metapneumovirus detection, can solve the problems that the avian metapneumovirus detection kit cannot detect poultry whole blood and cannot be applied to a variety of poultry, and achieves simple and fast sample processing , low cost, good sensitivity

Pending Publication Date: 2022-07-22
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] The technical problem to be solved by the present invention is to overcome the defects and deficiencies that the existing avian metapneumovirus detection kits cannot detect the collected poultry whole blood, nor can it be applied to various poultry, and provide a method that can detect poultry whole blood and can Avian Metapneumovirus Antibody Double Antigen Sandwich ELISA Detection Kit Suitable for Various Poultry and Subtypes

Method used

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  • Avian metapneumovirus antibody double-antigen sandwich method ELISA detection kit and application thereof
  • Avian metapneumovirus antibody double-antigen sandwich method ELISA detection kit and application thereof
  • Avian metapneumovirus antibody double-antigen sandwich method ELISA detection kit and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1A

[0056] Example 1 Preparation and purification of APMV / C-N protein

[0057] Preparation of reagents:

[0058] Buffer A: 1M Tris-HCl (pH 8.0) 50mL, 5M NaCl 100mL, glycerol 100mL, add water to make up to 1L, sterilize at 121°C for 20min, and store in a 4°C refrigerator.

[0059] Buffer B: imidazole 16.02g, 1M Tris-HCl (pH 8.0) 50mL, 5M NaCl 100mL, glycerol 100mL, add water to make up to 1L, sterilize at 121°C for 20min, and store in a refrigerator at 4°C.

[0060] Buffer C: imidazole 25.6g, 1M Tris-HCl (pH 8.0) 50mL, 5M NaCl 100mL, glycerol 100mL, add water to make up to 1L, sterilize at 121°C for 20min, and store in a refrigerator at 4°C.

[0061] Buffer D: imidazole 32.04g, 1M Tris-HCl (pH 8.0) 50mL, 5M NaCl 100mL, glycerol 100mL, add water to make up to 1L, sterilize at 121°C for 20min, and store in a refrigerator at 4°C.

[0062] Preparation process of AMPV / C-N protein:

[0063] (1) The pET-32a-AMPV / C-N prokaryotic expression vector plasmid constructed by BGI was introduce...

Embodiment 2

[0075] Example 2 HRP-AMPV / C-N horseradish peroxidase labeling

[0076] Take 10mg / mL horseradish peroxidase (HRP) solution 2mL, add 0.06M NaIO 4 2 mL of aqueous solution, oxidized at 4 °C for 30 min, added 2 mL of 0.4M ethylene glycol solution containing 22% NaCl, oxidized at 25 °C for 30 min, mixed with 24 mL of pre-cooled absolute ethanol, and centrifuged at 1500 rpm After 10 minutes, pour off the upper layer of liquid and place it on the test tube and filter paper. After the liquid is drained, add 8 mL of ultrapure water to dissolve the precipitate. After adding 4 mg of antigen solution (AMPV / C-N), adjust the pH to 9.0 with 0.5M CBS at pH 9.6. After standing at 4°C for 16-24h, add 10mg / mL KBH 4 Aqueous solution 0.2 mL, after standing for 2 h, add NaH 2 PO 4 Adjust pH to 7.0 to obtain HRP-AMPV / C-N with a protein concentration of 0.5 mg / mL.

Embodiment 3

[0077] Example 3 Establishment of avian metapneumovirus antibody double antigen sandwich ELISA detection kit

[0078] Preparation of ELISA plate:

[0079] 1. Coat AMPV / C-N protein on the ELISA reaction plate, the specific method is:

[0080] The AMPV / C-N protein was diluted to 0.6875mg / mL with 0.05M CBS pH 9.6, 100μL was added to each reaction well, coated at 4°C for 18h, and 120μL of blocking solution (10% sucrose solution) was added to each reaction well after washing the plate twice. , a mixed solution of 5% BSA and 5% fish gelatin, 1:1:1), closed at 37°C for 2 hours, then thrown off the blocking solution and dried the reaction plate in a 37°C oven to obtain a coated ELISA reaction plate.

[0081] Enzyme label preparation

[0082] 1. The HRP-AMPV / C-N obtained in Example 2 was diluted to 0.25 mg / L with 10% sucrose solution, and then 0.01% Royal blue (0.1 mL pigment per liter) was added to obtain the enzyme marker.

[0083] 3. Detection kit

[0084] Avian metapneumovirus an...

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Abstract

The invention belongs to the technical field of avian metapneumovirus detection, and particularly relates to an avian metapneumovirus antibody double-antigen sandwich method ELISA (enzyme-linked immuno sorbent assay) detection kit and application thereof. The kit provided by the invention can accurately and sensitively detect the avian metapneumovirus neutralizing antibody in poultry whole blood or serum so as to qualitatively judge a sample. The kit has the advantages of being high in sensitivity, good in sensitivity and capable of detecting various poultry and subtypes at the same time, sample treatment is simple and rapid, time consumption is short, and cost is low. The invention provides a good solution for on-site detection of large-batch avian metapneumovirus samples, and provides an important means for prevention and control of avian metapneumovirus.

Description

technical field [0001] The invention belongs to the technical field of avian metapneumovirus detection. More specifically, it relates to an avian metapneumovirus antibody double antigen sandwich ELISA detection kit and its application. Background technique [0002] Avian metapneumovirus (AMPV), also known as turkey rhinotracheitis virus (TRTV), earlier known as avian pneumovirus (APV), was later isolated from human , was reclassified as avian metapneumovirus. AMPV belongs to the family Paramyxoviridae, the subfamily Pneumoviridae, and the genus Metapneumovirus. The virus is an enveloped, non-segmented, single-stranded negative-stranded RNA virus. It is divided into A, B, and C according to the difference of the glycoprotein G protein. and D four subtypes. Under the counterstain electron microscope, AMPV is a highly pleomorphic particle with a diameter of 50-200 nm, usually spherical, and fibrils longer than 1000 nm in length. sudden. [0003] In addition to causing resp...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/53G01N33/531G01N33/569G01N21/31
CPCG01N33/53G01N33/531G01N33/56983G01N21/31
Inventor 杨金易毛志成苏晓娜龚馨梦张春枝黄庆锋
Owner SOUTH CHINA AGRI UNIV
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