Pathogen-associated molecular pattern immune detection chip, preparation method and application thereof

A molecular pattern, immune detection technology, applied in the fields of immune detection chips or microarrays, immunological detection, and preparation, can solve the problems of unintuitive results, large limitations, poor practicability, etc., to simplify the lack of detection technology and increase reliability. , the effect of fast response

Inactive Publication Date: 2010-11-24
INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The detection method based on SDS-PAGE is intuitive, but its throughput is low, and it is only suitable for insoluble PAMPs; the biomacromolecule interaction instrument detection method has high sensitivity and is easy to operate, but it needs to be equipped with an expensive biomacromolecule interaction instrument, which is not easy Realized; enzyme-linked immunosorbent assay is a commonly used detection method at present, but it has the disadvantages of unintuitive results and prone to false positives
These detection techniques have large limitations, low efficiency, time-consuming and poor practicability

Method used

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  • Pathogen-associated molecular pattern immune detection chip, preparation method and application thereof
  • Pathogen-associated molecular pattern immune detection chip, preparation method and application thereof

Examples

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Effect test

Embodiment 1

[0044] 1. Antibody acquisition and purification

[0045] Get healthy rat serum, use Amersham Phamacia Biotech's affinity chromatography column (HiTrap Protein G Sepharose Column) to purify rat Ig, use rat Ig as antigen, immunize purebred New Zealand white rabbits in a conventional method, and prepare serum by collecting blood. and chromatographic purification to obtain rabbit anti-rat Ig antibody.

[0046] 2. Chip structure design

[0047] The chip structure is as figure 1 As shown, including the crystal core polymer three-dimensional substrate (1), the crystal core polymer three-dimensional substrate (1) is fixed with two rows and five columns of a total of 10 6 × 5 PAMP microarrays (2), and the sample volume is 50-70nL, the spot diameter is 500-600μm. Each microarray is separated by a chip-specific fence or a Super PAP Pen scribe line (3). There is a labeling area (4) at one end of the slide.

[0048] 3. Preparation of PAMP and antibody samples

[0049] Dissolve variou...

Embodiment 2

[0055] Steps 1 and 2 are the same as in Example 1.

[0056] 3. Preparation of PAMP and antibody samples

[0057] Dissolve various PAMPs with phosphate buffered saline (PBS) containing 40% glycerol at pH 7.4 to make lipopolysaccharide (LPS), peptidoglycan (PGN), yeast glucan (Yeast glucan), Euglena β-1 , 3 glucan (β-1, 3glucan), mannan (mannan), lipoteichoic acid (LTA), Poly I:C concentration is 1.0mg / mL, CpG ODN concentration is 20μg / mL; dilute rabbit anti Rat Ig antibody, so that the concentration is 0.10mg.mL.

[0058] 4. Spot printing and fixation of samples

[0059] Spot the PAMP solution and antibody diluent on different regions of the agarose gel substrate with a spotting instrument, the spot volume is 50-70nL, and the spot diameter is 500-600μm. Each chip has two rows and five columns with a total of 10 6×5 subarrays. The samples in each subarray are the same. The first number is LPS, the second is PGN, the third is Yeast glucan, and the fourth is β-1, 3 glucan , No...

Embodiment 3

[0063] Steps 1, 2, and 3 are the same as in Example 2.

[0064] 4. Spot printing and fixation of samples

[0065] Spot the PAMP solution and antibody diluent on different regions of the agarose gel substrate with a spotting instrument, the spot volume is 50-70nL, and the spot diameter is 500-600μm. Each chip has two rows and five columns with a total of 10 6×5 subarrays. The samples in each subarray are the same. The first number is LPS, the second is PGN, the third is Yeast glucan, the fourth is β-1, 3glucan, No. 5 is mannan, No. 6 is LTA, No. 7 is CpG ODN, No. 8 is Poly I:C, and No. 9 is quality control point. The sample is rabbit anti-rat Ig antibody as positive control and fixed control. Ten No. is phosphate glycerol buffer as a negative control. 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, irradiate with 60mJ ultraviolet light for 10min, 20min, 30min, 40min,...

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Abstract

The invention relates to the field of immune detection of immunology and discloses a pathogen-associated molecular pattern immune detection chip, a preparation method and application thereof. The chip consists of a chip carrier, a pathogen-associated molecular pattern and an antibody, wherein the pathogen-associated molecular pattern and the antibody are fixed on the chip carrier. The chip carrier is a commercial crystal core high-molecular three-dimensional substrate. The preparation method of the pathogen-associated molecular pattern immune detection chip comprises the steps of the preparation of the pathogen-associated molecular pattern and the antibody microarray, the preparation of a specific polyclonal antibody for detection and the marking of fluorescein (Cy3) and also comprises the specific steps of simultaneously detecting the mutual action of a plurality of samples and the pathogen-associated molecular pattern by using the chip and carrying out condition optimization. The prepared pathogen-associated molecular pattern can be used for the research of the immune identification action mechanism and the research of mutual action of biological large molecules in medicine development.

Description

Technical field: [0001] The invention relates to immunological detection technology, in particular to an immunoassay chip or microarray for detecting the interaction between a pathogen-associated molecular pattern (PAMP) and a pattern recognition receptor (PRR), The preparation method and application thereof belong to the cross field of immunology, pathogenic microbiology and biochip technology. Background technique: [0002] Immune recognition is the basis for biological organisms to respond to foreign substances and achieve effective defense, and is a key link in the innate immune response. The host mediates and completes immune recognition through pattern recognition receptors (PRR), and the target molecules it recognizes are pathogen-associated molecular patterns (PAMPs) associated with the pathogenesis or survival of pathogenic microorganisms. PAMPs mainly include: bacterial lipopolysaccharides (LPS), peptidoglycan (PGN), lipoteichoic acids (LTA), CpG deoxyoligonucleot...

Claims

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Application Information

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IPC IPC(8): G01N33/53G01N33/531
Inventor 宋林生杨嘉龙邱丽梅王玲玲张峘
Owner INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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