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Non-diagnostic method for detecting poultry bursal disease virus by adopting surface ion resonance technology

A technical detection and diagnostic technology, applied in the field of biotechnology detection, can solve problems such as inability to screen antibodies or reference antigens, and achieve the effects of high sensitivity, strong specificity, and low operating costs

Inactive Publication Date: 2015-01-21
INSPECTION & QUARANTINE TECH CENT SHANDONG ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At the same time, most of the traditional immunoserological diagnostic methods need to label various antibodies, and cannot realize the screening of antibodies or reference antigens before large-scale application. An integrated diagnostic technology for high-throughput rapid screening and accurate identification of diagnostic methods before establishment and promotion

Method used

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  • Non-diagnostic method for detecting poultry bursal disease virus by adopting surface ion resonance technology
  • Non-diagnostic method for detecting poultry bursal disease virus by adopting surface ion resonance technology
  • Non-diagnostic method for detecting poultry bursal disease virus by adopting surface ion resonance technology

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] A non-diagnostic method for the detection of avian bursal virus by surface ion resonance, comprising the following steps:

[0020] 1. Conjugation of Mouse Anti-Antibody

[0021] (1) The sample does not need to be pretreated, and HBS is used as the buffer analysis buffer, and the mouse anti-antibody conjugated is immobilized on the CM5 chip. Set the coupling guidance mode: In the Surface Preparation window, select Immobilisation, Sensor Chip: CM5 and Amine Coupling methods at the same time, and set a blank reference channel that is not conjugated with mouse antibody.

[0022] (2) Equilibrate all reagents to a room temperature of 20-25°C, and set the flow rate of sample addition to 10 μL / min.

[0023] (3) Inject the sample, activate the surface of the chip with 110 μL NHS / EDC mixture in sequence, then add excess mouse anti-antibody to couple on the surface of the activated chip, after the coupling is completed, block with 60 μL Ethanolamine HCl to make the excess The re...

Embodiment 2

[0035] Embodiment 2 specificity test

[0036] Antigens of different strains were analyzed on the surface of the SPR chip. Pathogens, allantoic fluid and cell culture fluid were all filtered and loaded through a 0.22um microporous membrane, and there was no specific and significant binding reaction except for bursal virus, and no consistent with the above subtypes appeared in the SPR analysis pattern characteristic peaks. For the specificity test results of SPR, see figure 1 (The vertical axis is the RU response value of the instrument).

Embodiment 3

[0037] Embodiment 3 sensitivity analysis

[0038] Take three strains of bursal monoclonal antibody, and dilute the concentrations to 250, 125, 62.5, 31.25, 15.625 to 7.8125nmol / mL in sequence, and inject sequentially to obtain the binding and dissociation constant KD values ​​of the monoclonal antibodies (as shown in Table 1). And the combination rate and dissociation rate are measured by the formula, which is convenient for screening monoclonal antibodies with better immune response effect. by the formula (A is the analyte in the solution controlled by the flow system, the concentration of AB can be obtained by directly detecting the response signal RU of the instrument, and B is the molecule coupled on the sensor chip, which is equivalent to the maximum binding reaction theoretical value R max ) from which it can be deduced that

[0039]

[0040] Table 1 Kinetic data of three IBDV monoclonal antibodies

[0041]

[0042] Simultaneously capture the monoclonal antibody...

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Abstract

The invention discloses a non-diagnostic method for detecting a poultry bursal disease virus by adopting a surface ion resonance technology. The non-diagnostic method comprises the following steps: (1) coupling of an anti-mouse antibody, to be specific, coupling the anti-mouse antibody with a biosensor chip in a surface ion resonance system; (2) capturing of poultry bursal monoclonal antibodies and determining of capturing concentration; (3) detection of a sample, to be specific, enabling a sample to be detected to flow through a flowing pool of the biosensor chip in the surface ion resonance system, determining a signal difference value of the corresponding flowing pool before and after a mixture flowing through the chip, and determining whether the virus exists or not and the content of virus according to the signal difference value. According to the detection method, a positive response signal is avoided when other poultry source viruses are detected, so that the specificity is strong. The detection limit for recombinant proteins of poultry bursal disease virus reaches 0.625nmol / L, and the sensitivity is high.

Description

technical field [0001] The invention relates to a non-diagnostic method for detecting poultry bursal virus by surface ion resonance technology, which belongs to the field of biotechnology detection. Background technique [0002] Chicken infectious bursal disease is an acute, contagious disease caused by infectious bursal disease virus (IBDV). It is characterized by inflammation, necrosis, atrophy and severe damage of lymphocytes in the bursa of Fabricius, which causes immune dysfunction in chickens and interferes with the immune effects of various vaccines. At present, this disease is one of the three major diseases that endanger the poultry industry, and it is distributed worldwide. The disease causes immunosuppression of chicks, making sick chickens more susceptible to pathogens such as Escherichia coli, adenovirus, Salmonella, and chicken coccidia. Marek vaccine, poultry bursal virus vaccine and other vaccinations have reduced response ability, high incidence rate, almos...

Claims

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Application Information

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IPC IPC(8): G01N21/552
Inventor 孙涛曹丙蕾孙明君徐彪房保海王群
Owner INSPECTION & QUARANTINE TECH CENT SHANDONG ENTRY EXIT INSPECTION & QUARANTINE BUREAU