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Fish lymphocystis disease virus (LCDV) immunity detection chip and preparation method and application thereof

A lymphocyst virus, immunological detection technology, applied in the field of virology and biochip crossover, immunology, can solve the problems of sensitivity, specificity, accuracy can not be balanced, large limitations, etc., achieve low cost, increase reliability, The effect of increasing comparability

Inactive Publication Date: 2010-01-20
OCEAN UNIV OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these detection techniques have great limitations, and cannot balance sensitivity, specificity, and accuracy, and cannot be used for simultaneous parallel detection of multiple samples.

Method used

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  • Fish lymphocystis disease virus (LCDV) immunity detection chip and preparation method and application thereof
  • Fish lymphocystis disease virus (LCDV) immunity detection chip and preparation method and application thereof
  • Fish lymphocystis disease virus (LCDV) immunity detection chip and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] 1. Antibody preparation and purification

[0048] LCDV was extracted from surface tumors of flounder suffering from lymphocyst virus disease, purebred New Zealand white rabbits were immunized by conventional methods, blood was collected to prepare serum, and rabbit anti-LCDV antibodies were obtained.

[0049] Resuscitate and culture mouse hybridoma cell lines monoclonal antibody B and monoclonal antibody C, inject a certain number of hybridoma cells into the peritoneal cavity of mice to produce ascites, and obtain a large amount of high-concentration, high-specificity, high-titer mouse anti-LCDV monoclonal antibody.

[0050] Monoclonal antibody B and monoclonal antibody C purified by affinity chromatography were mixed, and purebred New Zealand white rabbits were immunized by conventional methods, blood was collected to prepare serum, and rabbit anti-mouse Ig antibodies were obtained.

[0051] The resulting antibody was purified using an affinity chromatography column (H...

Embodiment 2

[0065] Steps 1, 2, 3, 4, 5 are the same as in Example 1.

[0066] 6. Antibody Immobilization

[0067] ① Dilute the rabbit anti-LCDV antibody with pH 7.4 PBS buffer containing 50% glycerol to a concentration of 0.5 mg / mL; dilute the rabbit anti-mouse Ig antibody to a concentration of 0.1 mg / mL.

[0068] ②Use a spotting instrument to spot the diluted antibody solution on different areas of the surface of the modified glass slide, and the dot matrix distribution is as follows: figure 1 As shown, there are eight 4×4 subarrays in two rows and four columns per chip, and the samples in each subarray are the same. Among them, the samples in the first column are phosphate glycerol buffer as a negative control; the second, The samples in the third column are rabbit anti-LCDV antibodies; the samples in the fourth column are rabbit anti-mouse Ig antibodies as positive control and fixed control and other quality control points. Each subarray is separated by a chip-specific fence Super P...

Embodiment 3

[0071] The sandwich method was used to detect fish lymphocyst virus.

[0072] Steps 1, 2, 3, 4, 5, 6 are the same as in Example 2.

[0073] 7. Pathogen detection

[0074] ①Take the fish tissues to be tested (epidermal, gills, stomach or intestines, etc.), mix them with liquid A at a ratio of 1:10 (W / V), homogenate, freeze and thaw repeatedly for 3 to 4 times, and after ultrasonic crushing, Centrifuge at low temperature, 5000rpm×15min, take the supernatant as a test sample, to be tested;

[0075] ② Add the samples to be tested to different subarrays of the same carrier of the above-mentioned chip. The sample loading volume is 10 μL / array. One chip can detect eight samples at the same time. Pay attention to avoid liquid mixing between different subarrays. Incubate at 37°C with saturated humidity for 15min, 30min, 45min, 60min, 90min, and 120min, wash, add diluted C solution on the chip, 10μL / array, place at 37°C with saturated humidity for 15min, 30min, 45min, 60min, 90min, an...

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Abstract

The invention discloses a fish lymphocystis disease virus (LCDV) immunity detection chip, and the structure of the chip comprises a chip carrier, an agarose gel layer covered on the chip carrier and a plurality of antibody microarrays fixed on the agarose gel layer; wherein, special fences for chips or Super PAP Pen rulings are arranged between the microarrays for separating the microarrays. In the invention, a sandwich method is adopted to detect antigen, pathogenic antibody is fixed on a film support of the chip, the to-be-detected sample solution is prepared by using virus target organ of a sample to be detected, the to-be-detected sample solution and the chip fixed with pathogenic antibody are directly incubated, a specific monoclonal antibody (McAb) probe with a fluorescent mark is added, and then a CCD scanner is used for reading the result. The invention has the advantages of detecting lymphocystis disease virus in various samples or different tissues in the same sample simultaneously, which can be used for rapid, sensitive, and accurate detection on LCDV in marine-culturing fishes and quarantine on LCDV in imported-exported fishes.

Description

technical field [0001] The present invention relates to the improvement of pathogen detection technology for seawater cultured animals, in particular to an immune detection chip or microarray for detecting fish lymphocystis disease virus (LCDV) and its preparation method and application, which belong to immunology, Virology and biochip cross-technical field. Background technique [0002] Fish lymphocystosis is a chronic viral disease. Cauliflower-like cysts appear on the epidermis, fins and tail of diseased fish, affecting its commercial value or causing fish death. Lymphocystic disease is caused by Lymphocyst virus (LCDV), which belongs to the family Iridoviridae. Lymphocystic disease is endemic in a wide area and is distributed worldwide. It is known that more than 125 species of seawater, brackish water and brackish water fish in at least 42 families can be infected by LCDV. Since the first large-scale outbreak of flounder lymphatic cyst disease in my country in 1997, i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/569G01N33/577
CPCG01N33/56983
Inventor 绳秀珍徐晓丽战文斌
Owner OCEAN UNIV OF CHINA
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