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West nile virus monoclonal antibody and kit

A West Nile virus and monoclonal antibody technology, applied in the direction of antiviral immunoglobulin, biochemical equipment and methods, instruments, etc., can solve the problems of easy contamination of samples and operations, expensive consumables, false positives, etc. Achieve the effect of simple operation, rapid response and strong sensitivity

Inactive Publication Date: 2015-04-08
北京世纪元亨动物防疫技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this method requires expensive and professional equipment, especially real-time fluorescent RT-PCR. The consumables used are also expensive, and the operation requires professionally trained personnel to be competent. Moreover, samples and operations are prone to contamination and cause false positives.

Method used

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  • West nile virus monoclonal antibody and kit
  • West nile virus monoclonal antibody and kit
  • West nile virus monoclonal antibody and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Embodiment 1, West Nile virus monoclonal antibody hybridoma cell, the preparation of monoclonal antibody and polyclonal antibody

[0042] 1. Recombinant expression and purification of West Nile virus (WNV) envelope E protein.

[0043] In order to express the recombinant WNV E protein, the nucleic acid sequence of domain III of the E protein (the nucleic acid sequence encoding the 296-415 amino acids of the E protein) was artificially synthesized. Primers were designed according to the West Nile virus E protein gene. The upstream primer was 5'-AGCTCGAGCATGCAGTTGAAGGGAACAACCTATG-3', and the Xho I restriction site was introduced at the same time. The downstream primer was: 5'-TTTGGATCCTTACGCTCCTTTGAGGGTGG-3', and the BamHI restriction site was introduced. The target gene was cloned using the artificially synthesized WNV-E gene sequence as a template. PCR amplification program: 94°C for 4min, 94°C for 30s, 50°C for 30s, 72°C for 30s, 35 cycles, 72°C for 10min. The amplifi...

Embodiment 2

[0058] Embodiment 2: Titer determination of West Nile virus monoclonal antibody ascitic fluid

[0059] 1. Use the indirect ELISA method to measure the ascites titer of West Nile virus monoclonal antibody.

[0060] The specific method is:

[0061] 1) Coat the microtiter plate with purified West Nile virus E protein at a concentration of 1 μg / mL, freeze overnight at 4°C, and wash the microtiter plate.

[0062] 2) Seal the plate with phosphate buffer saline containing 2.5% BSA, wash the microplate at 37° C. for 30 min.

[0063] 3) Add ascites antibody solution diluted according to a certain ratio, 37°C, 2h. Wash the microtiter plate.

[0064] 4) Add alkaline phosphatase-labeled goat anti-mouse antibody diluted 1:1000, 37°C, 2h. Wash the microtiter plate.

[0065] 5) Add the substrate solution, at 37°C, read the OD value at 450nm with a microplate reader when the color is suitable (5-15min).

[0066] Such as Figure 4 As shown, the ascites antibody titer of Example 1 of the...

Embodiment 3

[0067] Embodiment 3: Ascites specificity determination of West Nile virus monoclonal antibody

[0068] 1. Identification of immunoglobulin classes and subclasses of monoclonal antibodies

[0069] 1. Follow the operating instructions of the Southern Biotech kit

[0070] (1) Coating: Dilute the antibody provided in the kit with carbonate buffer solution of pH 9.6 to a concentration of 5-10 μg / Ml100 μ / well, overnight at 4°C;

[0071] (2) Washing: wash the plate 3 times with PBS containing 0.05% Tween-20, and pat dry;

[0072] (3) Blocking: add 200 μL / well of PBST containing 1% bovine serum albumin, and incubate at 37° C. for 1 h to block the uncoated active site; wash as above;

[0073] (4) Sample addition: add 100 μL / well of the hybridoma culture supernatant of Example 1, place on a microtiter plate and incubate at 37° C. for 1 h; wash as above;

[0074] (5) Enzyme addition: Dilute alkaline phosphatase-labeled immunoglobulins, subclasses, heavy chains and light chains with PB...

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Abstract

The invention provides a west nile virus monoclonal antibody and a kit. A hybridomas cell strain secreting west nile virus monoclonal antibody is obtained, and has the preservation number of CGMCC No.8508. The obtained west nile virus monoclonal antibody possesses the advantages of good sensitivity, strong specificity, high titer and the like, and is applicable to identify whether a to-be detected virus is west nile virus and indentify whether a to-be detected sample contains west nile virus. The monoclonal antibody is applicable to prepare immunological diagnostic reagents of west nile virus, for example, an enzyme-linked diagnostic reagent, a colloidal gold immunochromatographic strip and the like.

Description

technical field [0001] The invention belongs to biological detection technology, and relates to a West Nile virus monoclonal antibody and a kit. Background technique [0002] West Nile Virus (WNV) is an arbovirus belonging to the Flaviviridae family and infects humans, birds, horses, poultry and other animals through mosquito bites. Before 1999, West Nile disease was mainly distributed in Africa, the Middle East, Europe, and West / Central Asia. After 1999, it was mainly distributed in North America. It is a viral infectious disease that seriously threatens human health and has become a global public health security problem. focus of attention. Severe cases of the disease can cause fatal meningitis or encephalitis. In 2012, 38 states in the United States reported human infection cases, at least 1118 cases were reported, and 41 deaths were reported. [0003] Birds are the natural storage hosts of West Nile virus, and the source of infection is mainly birds and infected anima...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/20C07K16/10G01N33/577G01N33/571C12R1/91
Inventor 陈西钊孙明张丽马永缨迟立超秦亚嫚
Owner 北京世纪元亨动物防疫技术有限公司
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