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Avian influenza virus H5N1 antibody molecule, and detection kit and application thereof

A technology for detecting kits and antibody molecules, applied in the field of biomedicine to achieve high specificity

Inactive Publication Date: 2014-03-05
广东省疾病预防控制中心
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the method of using antibodies to detect antigens has not yet been commercialized in China.

Method used

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  • Avian influenza virus H5N1 antibody molecule, and detection kit and application thereof
  • Avian influenza virus H5N1 antibody molecule, and detection kit and application thereof
  • Avian influenza virus H5N1 antibody molecule, and detection kit and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 2

[0023] Describe in detail below by embodiment.

[0024] Unless otherwise specified, the reagents used in the following examples are all analytically pure, and the coating solution is 0.1M NaHCO 3 Solution (pH8.6); PBS is phosphate buffered saline containing 0.1M Na 2 HPO 4 -NaH 2 PO 4 ; The blocking solution was PBS solution containing 3% BSA.

Embodiment 1

[0025] Example 1: Preparation of an antibody with specific binding activity to the A / H5N1 / Anhui / 1 / 2005 strain

[0026] 1. Screen the phage antibody library to obtain positive clones.

[0027] Dissolve the A / H5N1 / Anhui / 1 / 2005 strain to 100μg / ml with the coating solution, add 1ml of it on a small petri dish produced by Nunc Company (coating solution pH8.6NaHCO 3 ), placed in a humid box overnight at 4°C. Discard the coating solution, wash 3 times with PBS, add 3.5ml blocking solution (PBS containing 3% BSA), and incubate at room temperature for 2hr.

[0028] Discard the blocking solution, wash 3 times with PBS, add 500 μl Tommlison I+J library (purchased from MRC HGMP Resource Center, Cambridge, UK) and 3.5ml blocking solution, and incubate at room temperature for 2 hours.

[0029] Discard the supernatant and wash with PBS-0.1% TWEEN20. Wash 10 times in the first round, and 20 times in the 2nd and 3rd rounds.

[0030] Add 500 μl PBS-trypsin (add 50 μl of 10 mg / ml trypsin sto...

Embodiment 3

[0051] Embodiment 3: the specificity of the detection kit of embodiment 2

[0052] Use the coating solution to coat 50 μl of positive phage antibody molecules on a 96-well enzyme-linked plate, and place it in a humid box at 4°C overnight. Discard the coating solution, wash the wells 3 times with PBS, add 350 μl of blocking solution (PBS containing 3% BSA), and incubate at room temperature for 2 hours to block.

[0053] Discard the blocking solution, wash the wells 3 times with PBS, and add 10 μl of the following virus strain lysates to each well, including: H5N1 virus standard strains (gifted by the National Center for Disease Control and Prevention): Anhui / 1 / 2005 and A / Hubei / 2010, Enterovirus EV71, type I dengue virus, new type A H1N1, seasonal H1N1, seasonal H3N2 and type B virus and 90 μl blocking solution were incubated at room temperature for 1 hr. Discard the supernatant, wash the wells 3 times with PBS-0.1% TWEEN20, add 50 μl of positive phage antibody molecules and 50...

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Abstract

The invention discloses an avian influenza virus H5N1 antibody molecule, wherein the heavy chain variable region of the antibody molecule comprises sequences shown as SEQ NO: 1 and SEQ NO: 2, and the light chain variable region of the antibody molecule comprises sequences shown as SEQ NO: 3 and SEQ NO: 4. The antibody is applicable to preparation of a detection kit for detecting avian influenza virus H5N1.

Description

technical field [0001] This application belongs to the field of biomedicine, specifically, an antibody molecule containing a specific antibody molecular sequence pattern that can specifically bind to highly pathogenic avian influenza H5N1 virus, used as a detection reagent in the detection of highly pathogenic avian influenza H5N1 virus Application of ELISA detection method. Background technique [0002] The highly pathogenic avian influenza H5N1 virus belongs to the Orthomyxoviridae Influenzavirus genus. It is a single-stranded negative-sense RNA virus with an envelope structure. It contains 8 RNA segments and is known to encode 11 viral proteins. H5N1 virus is a highly mutated virus, and there are currently four representative strains prevalent in my country: A / H5N1 / Anhui / 1 / 2005, A / Guangdong-Shenzhen / 1 / 2011, A / Hubei / 1 / 2010and A / Chicken / Hong Kong / AP156 / 2008, which can specifically bind to the antibodies of these four representative strains, can be used as a reagent for det...

Claims

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Application Information

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IPC IPC(8): C07K16/10G01N33/569
Inventor 武婕柯昌文林锦炎张永慧曾宪鍫张宏斌
Owner 广东省疾病预防控制中心
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