Monoclonal antibodies against duck Tembusu virus, antigen detection kit and application

A duck Tembusu virus and monoclonal antibody technology, which is applied in the field of animal virology and immunology, can solve the problems of long detection period, cumbersome operation, and inability to detect, and achieve the effect of short detection time, high sensitivity and rapid detection

Active Publication Date: 2014-10-22
北京市动物疫病预防控制中心
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, the detection of duck new flavivirus mainly depends on the judgment of clinical symptoms, virus isolation and molecular biology methods, but these methods have long detection cycle, cumbersome operation, strict requirements on experimental conditions, and are not suitable for large-scale clinical applications. Scale detection
Because the infection of the duck Tembusu virus studied in the present invention belongs to a n

Method used

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  • Monoclonal antibodies against duck Tembusu virus, antigen detection kit and application
  • Monoclonal antibodies against duck Tembusu virus, antigen detection kit and application
  • Monoclonal antibodies against duck Tembusu virus, antigen detection kit and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1 Preparation and Identification of Duck Tembusu Virus Monoclonal Antibody

[0033] The present invention accidentally obtained three hybridoma cell lines with high titer and good specificity through a large number of screening work, which were respectively named duck Tembusu virus monoclonal antibody hybridoma cells 1D4, 3F2 and 3B4, and were released on August 26, 2013. It was sent to the Beijing China General Microorganism Culture Collection Management Center (CGMCC) for preservation (Address: No. 3, Datun Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Zip Code 100101), classified as hybridoma cells, and its preservation The numbers are CGMCC NO:8104, CGMCC NO:8107, and CGMCC NO:8106. The acquisition and detection of the three monoclonal antibodies are described in detail below.

[0034] . Preparation of Duck Tembusu Virus Antigen

[0035] 1.1 Preparation of virus

[0036] The DTMUV-JXSP strain virus was prop...

Embodiment 2

[0060] Example 2 Establishment of Double Antibody Sandwich ELISA Antigen Detection Method for Duck Tembusu Virus

[0061] 1. Establishment of double-antibody sandwich ELISA antigen detection method for duck Tembusu virus

[0062] 1) Coating: dilute anti-DTMUV monoclonal antibody 100 μL / well with 0.05mol / LPH9.6 carbonate buffer, and coat overnight at 4°C;

[0063] 2) Washing: Dry the liquid in the wells of the plate, wash with washing liquid four times, each time for 4 minutes;

[0064] 3) Blocking: add blocking solution, i.e. sample diluent, 200 μL / well, block at 37°C for 2 hours;

[0065] 4) Washing: Dry the liquid in the wells of the plate, and wash with washing liquid four times, each time for 4 minutes;

[0066] 5) Add antigen: Antigen is properly diluted with sample diluent, 100 μL / well, incubated at 37°C for 1 hour;

[0067] 6) Washing: Dry the liquid in the wells of the plate, and wash with washing liquid four times, each time for 4 minutes;

[0068] 7) Add enzy...

Embodiment 3

[0095] Example 3 Assembly of Duck Tembusu Virus Double Antibody Sandwich ELISA Antigen Detection Kit

[0096] 1. ELISA kit assembly:

[0097] 96-well ELISA plate coated with anti-Duck Tembusu virus monoclonal antibody 3B4;

[0098] Standard positive control: Tambusu virus DTMUV antigen;

[0099] Standard negative control: BHK cell protein;

[0100] (4) Antibody 3F2 labeled with horseradish peroxidase;

[0101] ⑸ Washing liquid: NaCl 0.8g, KH 2 PO 4 0.27g, Na 2 HPO 4 . 12H 2 O 1.42g, KCl 0.2g, Tween20 0.5mL, add double distilled water to 1000mL;

[0102] ⑹ Sample diluent (blocking solution): NaCl 0.8g, KH 2 PO 4 0.27g, Na 2 HPO 4 . 12H 2 O 1.42g, KCl 0.2g, BSA 10g, add double distilled water to 1000ml;

[0103] ⑺ Substrate solution A: 3, 3’, 5’, 5-tetramethylbenzidine diamine (TMB) 200mg, absolute ethanol 100ml, add double distilled water to 1000ml;

[0104] ⑻ Substrate solution B: Na 2 HPO 4 14.6g, citric acid 9.3g, 0.75% hydrogen peroxide ur...

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Abstract

The invention provides monoclonal antibodies against the duck Tembusu virus, an antigen detection kit and application. According to the invention, the DTMUV-JXSP cell strain of the duck Tembusu virus is used as immunogen for preparation of the monoclonal antibodies which include monoclonal antibodies respectively secreted by hybridoma cells with respective accession numbers of CGMCC No. 8104, CGMCC No. 8107 and CGMCC No. 8106; the monoclonal antibody 3B4 secreted by the hybridoma cell with the accession number of CGMCC No. 8106 is used as a primary antibody coated ELISA plate, the monoclonal antibody 3F2 secreted by the hybridoma cell with the accession number of CGMCC No. 8107 is subjected to enzyme labeling and used as an ELISA secondary antibody, so the double-antibody sandwiched detection kit is established and the kit has good sensitivity and specificity. According to the invention, effective means are provided for large-scale clinical detection of the duck Tembusu virus.

Description

technical field [0001] The invention relates to the technical field of animal virology and immunology. Specifically relates to the preparation of a duck tembusu virus-specific monoclonal antibody, the preparation of a duck tembusu virus antigen detection ELISA kit containing the monoclonal antibody, and its application in duck tembusu virus antigen detection. Background technique [0002] Since April 2010, diseases characterized by a sharp drop in the egg production of laying ducks have appeared in Zhejiang, Jiangsu, Shandong, Hebei, Beijing and other provinces and cities in my country. The main clinical symptom of the disease is that laying ducks of all ages suddenly appear group egg production decline, or even complete production. Laying ducks have a long-term (40-60 days) low egg production rate after the onset of the disease, and the hatching rate and fertilization rate of the eggs produced after recovery decrease. Due to factors such as secondary infection, or drug tr...

Claims

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Application Information

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IPC IPC(8): C07K16/10C12N5/20G01N33/577G01N33/569C12R1/91
Inventor 冯小宇
Owner 北京市动物疫病预防控制中心
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