On-site detection immuno-chip and preparation method thereof and application

An immune chip and on-site detection technology, which is applied in the interdisciplinary field of immunology, biochip and diagnostic reagents, can solve the problems of difficult popularization and application of conventional protein chips

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

Combining immunoenzyme technology with protein chip technology, a kind of immune chip for on-site detection that can be used for simultaneous parallel detection of multiple samples is prepared, the test resu

Method used

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  • On-site detection immuno-chip and preparation method thereof and application
  • On-site detection immuno-chip and preparation method thereof and application
  • On-site detection immuno-chip and preparation method thereof and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] 1. Antibody acquisition and purification

[0064] Extract WSSV from gills and other target organs of prawns suffering from white spot virus disease, extract LCDV from surface tumors of flounder suffering from lymphocyst virus disease, immunize purebred New Zealand white rabbits by conventional methods, and collect blood to prepare serum to obtain rabbit anti-WSSV , Rabbit anti-LCDV antibody.

[0065] Resuscitate and culture mouse hybridoma cell lines (WSSV monoclonal antibody D, E, LCDV monoclonal antibody B, C), inject mice into the peritoneal cavity to produce ascites, and obtain mouse anti-WSSV monoclonal antibody and mouse anti-LCDV monoclonal antibody.

[0066] Take the mouse anti-WSSV monoclonal antibody (mixture of monoclonal antibody E and monoclonal antibody D) and mouse anti-LCDV monoclonal antibody (mixture of monoclonal antibody B and monoclonal antibody C) purified by affinity chromatography, and immunize purebred New Zealand white rabbits by conventional m...

Embodiment 2

[0071] Preparation of slides with seven different modification methods and comparison of fixation effects.

[0072] 1. Commercially available glass slides were washed with strong alkali and concentrated sulfuric acid, soaked in ultrapure water overnight, and modified by the following methods.

[0073](1) Preparation of APES-modified glass slides: APES is ready-to-use and ready-to-use. Put the cleaned slides into APES diluted with 1:50 acetone, stay for 20-30s, take it out and pause for a while, then put it into pure acetone solution to remove unbound APES, and immediately immerse in pure water for 2-5 minutes Rinse, store in a dust-free place away from light, and dry in an oven at 80°C on a slide rack.

[0074] (2) Preparation of poly-lysine-modified glass slides: put the cleaned and dried glass slides into poly-lysine solution diluted with deionized water at a ratio of 1:10, shake on a shaker, double Rinse with steamed water, soak for 5 minutes, bake in an oven at 60°C for ...

Embodiment 3

[0084] Antibody microarrays were prepared using the above-mentioned agarose-modified slide glass as a substrate.

[0085] 1. Chip structure design

[0086] The chip structure is as Figure 4 As shown, it includes the chip carrier (1), the agarose gel layer (2) covered on the chip carrier (1), and the agarose gel layer (2) is fixed with two rows and four columns, a total of eight 4×4 Antibody microarray (3), the sample volume is 50-70 nl, and the spot diameter is 500-600 μm. Each microarray is separated by a chip-specific fence or a Super PAP Pen scribe line (4). The chip carrier is a standard glass slide, and a label area (5) can be reserved at one end of the glass slide.

[0087] 2. Selection of the optimal concentration of capture antibody

[0088] (1) Dilute the capture antibody (rabbit anti-LCDV antibody or rabbit anti-WSSV antibody) with PBS buffer solution containing 50% glycerol at pH 7.4, so that the concentration is 1, 0.5, 0.1, 0.05, 0.01, 0.005, 0.001, 0.0005 m...

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Abstract

The invention discloses an on-site detection immuno-chip, 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 double antibody sandwich principle is adopted to detect the antigen to carry out the dot matrix of corresponding capture antibody, positive control and negative control on the solid phase carrier simultaneously; the antibody protein is connected with the solid phase carrier through the covalent bond and physical adsorption; the sample liquid to be detected and the chip are directly incubated; the antigen to be detected in the samples combine with the corresponding antibody which is fixed on the chip; a specific monoclonal antibody probe marked by horse radish peroxidase is added and the macroscopic detection results can be obtained after the coloration of substrates. The invention can detect viruses in multiple aquatic animal samples and the results are macroscopic, thus being applicable to rapid and accurate detection of viruses of aquatic animals in breeding production.

Description

technical field [0001] The invention relates to an on-site detection immune chip or microarray and a preparation method thereof, which is an improvement of pathogen detection technology, and specifically relates to a method for on-site detection of one or several types of The invention relates to a method and application of an immune chip for aquatic animal viruses, belonging to the interdisciplinary technical field of immunology, biological chips and diagnostic reagents. Background technique [0002] Viral diseases are the most difficult to prevent and treat among aquaculture animal diseases. Due to the specific aquatic environment of aquatic animals and the unique biological characteristics of viruses, the prevention and treatment of viral diseases are more difficult than those of other terrestrial animals. . At present, there is no effective method for treating viral diseases, so the prevention of viral infection is particularly important, and the accurate detection and ...

Claims

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Application Information

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IPC IPC(8): G01N33/552G01N33/569G01N33/577G01N33/532G01N33/52
Inventor 绳秀珍徐晓丽战文斌
Owner OCEAN UNIV OF CHINA
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