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Colloidal gold immunochromatograohic assay detection test strip based on NDV (Newcastle Disease Virus) hemagglutinin protein monoclonal antibody

A technology of hemagglutinin protein and monoclonal antibody, which is applied in anti-virus immunoglobulin, analytical materials, measuring devices, etc., can solve the problems of inconvenience for popularization and use in grass-roots farms, cumbersome and time-consuming methods, etc.

Inactive Publication Date: 2014-07-23
INST OF ANIMAL SCI & VETERINARY MEDICINE SHANDONG ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In recent years, molecular biology techniques such as RT-PCR, fluorescent quantitative PCR, nucleic acid diagnostic technology, and restriction endonuclease analysis have also been widely used in the diagnosis of ND. It is inconvenient to promote the use of advanced equipment in grass-roots farms

Method used

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  • Colloidal gold immunochromatograohic assay detection test strip based on NDV (Newcastle Disease Virus) hemagglutinin protein monoclonal antibody
  • Colloidal gold immunochromatograohic assay detection test strip based on NDV (Newcastle Disease Virus) hemagglutinin protein monoclonal antibody
  • Colloidal gold immunochromatograohic assay detection test strip based on NDV (Newcastle Disease Virus) hemagglutinin protein monoclonal antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0074] Example 1 Preparation of monoclonal antibody hybridoma cell line

[0075] Virus purification: Inoculate 9-10-day-old chicken embryos with NDV LaSota standard virus, harvest the allantoic fluid of the dead chicken embryos, freeze and thaw the allantoic fluid once, centrifuge at 15,000 rpm / min for 1 h, collect the supernatant and then pass 30,000 rpm / min The high-speed centrifuge was centrifuged for 2.5h, the supernatant was removed, the precipitate was suspended with PBS (pH7.4), and centrifuged at 35,000rpm / min for 2min with a sucrose density gradient to harvest the purified NDV LaSota virus;

[0076] Using the purified NDV LaSota virus as the immunogen, 6 Balb / c mice aged 6 to 8 weeks were immunized with a dose of 50ug each time. The immunization method was intraperitoneal injection, and immunized four times in total; The interval is 2 weeks, the protein is emulsified with Freund's complete adjuvant at a volume ratio of 1:1 for the first vaccination, and the protein is...

Embodiment 2

[0081] The acquisition, identification and purification of monoclonal antibody in Example 2

[0082] 1. Preparation of monoclonal antibody ascites

[0083]8-10-week-old Balb / c mice were intraperitoneally injected with 0.5 mL of sterile liquid paraffin oil, and 500,000 positive hybridoma cells in the logarithmic growth phase were intraperitoneally injected 7 days later; Swell, extract the ascites and centrifuge and take the supernatant to obtain the monoclonal antibody prepared by the positive hybridoma cell C3B3;

[0084] Fat and blood cells in the ascites can be removed by centrifugation, and a sufficient amount of monoclonal antibodies can be obtained from the ascites for subsequent identification and preparation;

[0085] 2. Characterization of monoclonal antibodies

[0086] 2.1 The specific identification of monoclonal antibody is coated with SPF chicken embryo allantoic fluid, Newcastle disease virus, avian influenza virus, chicken infectious bronchitis virus, infectiou...

Embodiment 3

[0099] The preparation of embodiment 3 test strips

[0100] 1. Preparation of Colloidal Gold

[0101] Using the improved trisodium citrate reduction method to prepare colloidal gold with different diameters, the specific steps are as follows: (all involved are weight percentages)

[0102] Take 100 mL of 0.01% chloroauric acid and place it in a conical flask and heat it on a constant temperature heating magnetic stirrer until it boils;

[0103] Accurately absorb 2.0mL of 1% trisodium citrate, quickly add it to the conical flask, shake it evenly, immediately place it on a constant temperature heating magnetic stirrer to continue boiling, and control the rotation speed at 100r / min until the golden yellow chloroauric acid becomes After the red color is boiled for 15 minutes, it is taken out, cooled and filtered, and stored at low temperature to maintain the colloidal state. The appearance of colloidal gold prepared by this method is clear and transparent orange-red, and there ar...

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Abstract

The invention relates to the field of bioengineering, and particularly relates to a newly prepared hybridoma cell strain for resisting NDV (Newcastle Disease Virus)hemagglutinin protein. A monoclonal antibody with a broad spectrum neutralization effect on NDV is obtained by using the cell strain, and an immune colloidal gold test strip for quickly detecting a newcastle disease virus is developed by using the antibody. By adopting the test paper disclosed by the invention, the result can be obtained within 5 minutes by sampling once, and the test paper has the characteristics of being simple and rapid, specific and sensitive. A more practical tool and method are provided for rapid diagnosis of a newcastle disease by development of the test strip, and the colloidal gold immunochromatograohic assay detection test strip is applicable to primary veterinary stations and small and medium-sized farms.

Description

technical field [0001] The invention relates to diagnostic reagents in the field of veterinary biological products, and specifically provides a monoclonal antibody with broad-spectrum neutralizing activity, and NDV colloidal gold immunochromatography technology and test strips established with the antibody. Background technique [0002] Newcastle disease is a highly contagious and contagious disease caused by Newcastle disease virus, which can cause a large number of poultry deaths or a sharp decline in production performance, and cause huge economic losses to the world poultry industry. Scholars at home and abroad have carried out a lot of research on the diagnostic methods of Newcastle disease. The traditional diagnostic methods include virus isolation, ELISA, agar diffusion test, neutralization test, hemagglutination and hemagglutination inhibition test. In recent years, molecular biology techniques such as RT-PCR, fluorescence quantitative PCR, nucleic acid diagnosis tec...

Claims

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Application Information

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IPC IPC(8): C12N5/20C07K16/10G01N33/577G01N33/569
Inventor 杨少华许传田胡北侠张琳黄庆华伊惠张秀美
Owner INST OF ANIMAL SCI & VETERINARY MEDICINE SHANDONG ACADEMY OF AGRI SCI
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