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Immunity detection chip of prawn white spot syndrome virus (WSSV) and preparation method thereof and application

An immunodetection and vitiligo technology is applied in the field of immunodetection chip or microarray, preparation, and improvement of marine aquaculture animal pathogen detection technology, which can solve the problems of prone to false positives, complicated operation, poor practicability, etc. Simple, responsive effects

Active Publication Date: 2012-09-26
OCEAN UNIV OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Electron microscopy is reliable but time-consuming; PCR method is highly sensitive and can be used for pathogen detection in asymptomatic aquatic animals, but it is prone to contamination and false positives; DNA probe in situ hybridization method has high specificity and sensitivity, but, The use of isotope-labeled nucleic acid probes for molecular hybridization is radioactive and complicated to operate, while non-radioactive-labeled probes are often less sensitive; enzyme-linked immunosorbent assay and dot immunoblotting are currently commonly used detection methods, but Endogenous enzymes will interfere with its detection results, resulting in false positives
These detection techniques have certain limitations, such as low efficiency, long time-consuming, and poor practicability.

Method used

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  • Immunity detection chip of prawn white spot syndrome virus (WSSV) and preparation method thereof and application
  • Immunity detection chip of prawn white spot syndrome virus (WSSV) and preparation method thereof and application
  • Immunity detection chip of prawn white spot syndrome virus (WSSV) and preparation method thereof and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] 1. Antibody acquisition and purification

[0054] WSSV was extracted from the target organ of white spot virus diseased prawns, and purebred New Zealand white rabbits were immunized by conventional methods, blood was collected to prepare serum, and rabbit anti-WSSV antibodies were obtained.

[0055] Mouse hybridoma cell lines monoclonal antibody D and monoclonal antibody E were recovered and cultured, and mice were injected into the peritoneal cavity to produce ascites, and a large amount of high-titer, high-specificity mouse anti-WSSV monoclonal antibody was obtained.

[0056] Take monoclonal antibody D and monoclonal antibody E purified by affinity chromatography, mix them, and routinely immunize purebred New Zealand white rabbits, collect blood to prepare serum, and obtain rabbit anti-mouse Ig antibody.

[0057] The resulting antibody was purified using an affinity chromatography column (HiTrap Protein G SepharoseColumn) from Amersham Phamacia Biotech.

[0058] 2. P...

Embodiment 2

[0071] Steps 1, 2, 3, and 4 are the same as in Example 1.

[0072] 5. Preparation of Agarose Gel Substrates

[0073] Prepare a 1.2% agarose solution, boil it in a microwave oven for 3 minutes to dissolve it completely, spread 2mL of the agarose solution on a clean slide treated with affinity silane preheated at 60°C; after the agarose solidifies, dry the slide overnight at 37°C ; Use 0.02mol / L NaIO before use 4 The solution was activated at room temperature for 30 minutes, rinsed thoroughly with ultrapure water three times, blown dry with nitrogen flow, and stored in a dry place at room temperature.

[0074] 6. Antibody Immobilization

[0075] ①Dilute rabbit anti-WSSV antibody and rabbit anti-mouse Ig antibody with PBS buffer solution containing 10%, 20%, 30%, 40%, 50%, and 60% glycerol at pH 7.4 to make the concentration 0.1mg / mL .

[0076] ② Spot the antibody dilution on different areas of the surface of the glass slide with a spotting instrument. Each chip has two rows ...

Embodiment 3

[0079] Steps 1, 2, 3, 4, 5 are the same as in Example 2.

[0080] 6. Antibody Immobilization

[0081] ①Dilute the rabbit anti-WSSV antibody with PBS buffer solution containing 50% glycerol at pH 7.4 to make the concentration 0.5, 0.1, 0.05, 0.01, 0.005, 0.001, 0.0005, 0.0001 mg / mL.

[0082]②Use a spotting instrument to spot different concentrations of antibody dilutions on different areas of the slide surface. Each chip has two rows and four columns with a total of eight 4×4 subarrays, and each subarray contains different concentrations of rabbit anti-WSSV antibodies. Each subarray is separated by a chip-specific fence or a Super PAP Pen to form an independent reaction unit. Place in saturated humidity at 37°C for 2 hours, wash and dry.

[0083] ③ Add 3% bovine serum albumin dropwise to the area on the slide where the antibody is spotted to block, place at 37°C in saturated humidity for 1 hour, wash, dry, and seal and store at 4°C to obtain the prawn white spot virus immunoa...

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Abstract

The invention discloses an immunity detection chip of prawn white spot syndrome virus (WSSV), comprising a chip carrier and an agarose gel layer which is paved on the chip carrier, wherein, the agarose gel layer is fixed with a plurality of antibody microarrays of 4*4, and chip-dedicated fences or Super PAP Pen scribings are used for separating the microarrays from each other. The invention comprises the following steps: adopting the sandwich method to detect the antigen; fixing the pathogenic polyclonal antibody (PcAb) on the chip slice base; taking the target organ tissue of the individual to be detected to prepare to-be-detected sample liquid; incubating directly the to-be-detected sample liquid with the chip which is fixed with PcAb to capture the antigen and lead the antigen to combine on the chip; adding a specific monoclonal antibody probe marked by fluorescence; and reading results through a CCD chip scanner. The invention has the advantage of detecting white spot syndrome virus (WSSV) in multiple samples simultaneously, and is applicable to the rapid and accurate detection of white spot syndrome virus (WSSV) of prawns / crabs in breeding production and the quarantine inspection of WSSV in import and export prawns / crabs.

Description

technical field [0001] The present invention relates to the improvement of pathogen detection technology of seawater cultured animals, in particular to an immunodetection chip or microarray, preparation method and application thereof for detecting white spot syndrome virus (WSSV) of prawns, which belong to immunology, The intersection of virology and biochip technology. Background technique [0002] Diseases seriously threaten the development of the shrimp farming industry. Various diseases have caused great losses to the shrimp farming. However, irrational use of drugs in disease control has caused drug resistance, drug residues, and diffusion in the environment, seriously affecting food safety and Environmental public health, therefore, early and accurate detection and diagnosis of cultured shrimp diseases is particularly important for disease prevention and control. White spot disease virus (WSSV) caused by white spot disease virus (WSSV) is one of the serious diseases i...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/569G01N33/577G01N33/552G01N33/532G01N21/64
Inventor 绳秀珍徐晓丽战文斌
Owner OCEAN UNIV OF CHINA
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