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Pigeon Newcastle disease virus monoclonal antibody, and applications thereof in preparation of diagnosis and detection kit

A technology of Newcastle disease virus and monoclonal antibody, applied in the field of agricultural biology, can solve the problems of poor sensitivity, large error, lack of reliability, etc., and achieve the effect of simple operation

Inactive Publication Date: 2018-11-30
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this method also has obvious shortcomings, such as poor sensitivity, large error, and small amount of samples detected each time, etc., lacks certain reliability, and requires a lot of manpower when testing a large number of samples.

Method used

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  • Pigeon Newcastle disease virus monoclonal antibody, and applications thereof in preparation of diagnosis and detection kit
  • Pigeon Newcastle disease virus monoclonal antibody, and applications thereof in preparation of diagnosis and detection kit
  • Pigeon Newcastle disease virus monoclonal antibody, and applications thereof in preparation of diagnosis and detection kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Obtaining of embodiment 1 hybridoma cells and preparation of pigeon Newcastle disease virus monoclonal antibody

[0048] 1. Pigeon Newcastle Disease Virus Propagation and Purification

[0049] (1) Collect nasal swabs or homogenate of glandular stomach, duodenum, brain and other tissues of dead pigeons in Xincheng, filter and sterilize through a filter with a pore size of 0.22 μm, and inoculate the 9-day-old through the allantoic cavity SPF chicken embryos, 100 μL / piece, humidity 65% ​​to 70%, incubated in an incubator at 37°C, observed the death of chicken embryos, discarded chicken embryos that died within 24 hours, and collected the allantoic fluid of chicken embryos that died within 60 to 72 hours to obtain Amplified pigeon Newcastle disease virus;

[0050] (2) collect pigeon Newcastle disease virus, inactivate with 0.03% formalin at 4 ℃, inactivate pigeon Newcastle disease virus through HA detection, determine the titer of inactivated pigeon Newcastle disease virus...

Embodiment 2

[0103] Embodiment 2, the colloidal gold diagnostic kit of pigeon Newcastle disease virus

[0104] 1. Colloidal gold labeled with pigeon Newcastle disease virus monoclonal antibody

[0105] (1) Determine the most suitable pH value of the colloidal gold-labeled antibody:

[0106] a. Sequentially take 1mL nano-gold solution into nine 1.5mL centrifuge tubes, and then sequentially add 1-9μL 0.1mol / L K 2 CO 3 The solution was added to 9 centrifuge tubes to adjust the pH value of the nano-gold solution, and then 20.0 μL of 1.0 mg / mL 3G1 antibody was sequentially added to each of the nano-gold solutions with different pH values, mixed gently, and allowed to stand at room temperature for 2 h. Observe the phenomenon.

[0107] b. After adding the antibody, centrifuge the mixed gold nano solution at 12,000rpm for 35min with no change in color and no coagulation, collect the supernatant, and measure the OD value of the supernatant by indirect ELISA method, and measure it with K 2 CO 3...

Embodiment 3

[0148] Embodiment 3, detect the competition ELISA kit of Newcastle disease antibody

[0149] 1. Preparation method of enzyme plate

[0150] Use 0.05M pH9.6 carbonate buffer as coating solution, dilute Newcastle disease virus, add 100μl / well into the reaction plate, coat overnight at 4°C, discard the coating solution the next day, and add 200μl Add 2% BSA blocking solution to each well, incubate at 37°C for 1 hour, wash and dry, dry at room temperature, put into a packaging bag, add desiccant, and store in vacuum to obtain an ELISA plate, which is the coated inactivated Newcastle disease virus standard ELISA plate for hemagglutination inhibitory antigen.

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Abstract

The invention discloses a pigeon Newcastle disease virus monoclonal antibody, and applications thereof in preparation of a diagnosis and detection kit. The pigeon Newcastle disease virus monoclonal antibody is high in sensitivity, specificity, and titer. The colloidal gold diagnostic kit is used for detecting whether the liver, spleen, lung, brain, or heat tissue homogenate of infected pigeons orpigeons newly died of illness, and oral and nasal cavity secretion contains pigeon Newcastle disease virus, so that auxiliary diagnosis of pigeon Newcastle disease is realized. Response is rapid; thewhole operation time is only 15min; no professional is required; popularization is convenient. The competitive ELISA kit used for detecting the pigeon Newcastle disease virus antibody is capable of representing the titer of the pigeon Newcastle disease virus antibody in serum through detecting the inhibition effect of serum antibody in a detected sample on horseradish peroxidase labelled anti-pigeon Newcastle disease virus monoclonal antibody combined pigeon Newcastle disease virus antigen, operation of the kit is simple and reliable.

Description

technical field [0001] The invention relates to the field of agricultural biotechnology, and refers to a pigeon Newcastle disease virus monoclonal antibody and its application in the preparation of diagnostic and detection kits, specifically anti-pigeon Newcastle disease virus monoclonal antibody and colloidal gold for pigeon Newcastle disease virus Diagnostic kits and competition ELISA kits for detection of Newcastle disease antibodies. It can be used for rapid on-site diagnosis of pigeon Newcastle disease virus and quantitative detection of antibodies after Newcastle disease immunization. Background technique [0002] Newcastle disease (ND) is an acute, highly contagious disease mainly infecting poultry caused by a virulent strain of Newcastle disease virus (NDV). Newcastle disease can not only cause serious harm to poultry, but also have a serious impact on international trade. The World Organization for Animal Health (OIE) lists Newcastle disease as a legally notifiabl...

Claims

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Application Information

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IPC IPC(8): G01N33/577G01N33/535
CPCG01N33/577G01N33/535
Inventor 张安定韩丽任海洋万颖李治
Owner HUAZHONG AGRI UNIV
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