Lectin-macromolecule carrier coupling compound for separating glycosylated exosome in clinical sample

A macromolecular carrier, lectin technology, applied in the preparation of test samples, separation methods, liquid separation of solid adsorbents, etc., can solve the problems of large demand for samples, low separation efficiency, large steric hindrance, etc. High separation efficiency, good repeatability and complete morphology

Active Publication Date: 2020-06-09
北京尧景基因技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them: ultracentrifugation or gradient density centrifugation is based on the small difference in the density of exosomes and other biological components to separate, this method requires special equipment, the demand for samples is relatively large, it takes a long time, and because the characteristics of different clinical samples are different , the stability of the separation effect using this method is poor, and further ultracentrifugation may easily cause vesicle damage and affect subsequent experiments; the principle of the immunomagnetic bead method is that specific proteins on the surface of exosomes, such as CD9 / CD63 / CD81, etc. The immunomagnetic beads with corresponding antibodies are combined for magnetic separation. However, due to the small size of the immunomagnetic beads, the particle size is at the nanometer level and less than or equal to the diameter of the exosomes, so the space for the combination of the immunomagnetic beads and the exosomes is Larger resistance, insufficient binding of exosomes, and low separation efficiency
In addition, the exosomes obtained by immunomagnetic bead separation are eluted with acidic eluent, resulting in incomplete morphology of exosomes.

Method used

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  • Lectin-macromolecule carrier coupling compound for separating glycosylated exosome in clinical sample
  • Lectin-macromolecule carrier coupling compound for separating glycosylated exosome in clinical sample
  • Lectin-macromolecule carrier coupling compound for separating glycosylated exosome in clinical sample

Examples

Experimental program
Comparison scheme
Effect test

Embodiment example 1

[0068] Implementation Case 1 Preparation of Lectin-Macromolecule Carrier Coupling Complex

[0069] The lectin-macromolecular carrier coupling complex includes: a macromolecular carrier; and a lectin coupled outside the macromolecular carrier, which is prepared by coupling a lectin and a macromolecular carrier under specific conditions, and is mainly used for The glycosylated exosomes in the sample were separated, and the glycosylated exosomes obtained after separation were complete in shape. in:

[0070] The selection of lectins mainly includes: Jacalin, peanut agglutinin (PNA), pea agglutinin (VVA and / or VVL), concanavalin bean agglutinin (ConA), lentil agglutinin (LCA), wheat germ Any one of WGA, soybean agglutinin (SBA), kidney bean agglutinin (PVL), snail agglutinin (HAA and / or HPA), or a combination of two or more lectins, the main reason is : The combination of different lectins can realize the separation of various types of glycosylated exosomes, such as: the separati...

Embodiment example 2

[0083] Implementation Case 2 Preparation of Glycosylated Exosome Isolation Composition

[0084] The present invention provides a composition for separating glycosylated exosomes, which specifically includes: the affinity adsorption centrifuge tube filled with LCA-agarose microsphere preservation solution obtained in Example 1, that is, glycosylated exosomes The separation device, cleaning solution, and eluent are packaged independently, and exist in a set or kit at the same time.

[0085] The above cleaning solution is a metal salt ion-free cleaning buffer or purified water, which can be selected as a metal salt ion-free cleaning buffer; for example: the main component includes 10-200mM metal salt ion-free TRIS-HCl buffer, and its pH value is 7.6 ±0.2. In this implementation case, the cleaning solution is a metal salt ion-free cleaning buffer, and its main component includes 100 mM metal salt ion-free TRIS-HCl buffer solution with a pH value of 7.6±0.2, which is used for clea...

Embodiment example 3

[0087] Implementation Case 3 Method of Using the Glycosylated Exosome Isolation Composition

[0088] The present invention also provides a method for separating glycosylated exosomes, including the experimental steps of using the above-mentioned glycosylated exosome separation composition, including:

[0089] 1. Preparation before experiment

[0090] Self-provided equipment or equipment: centrifuge, used for the centrifugation step in the process of separating glycosylated exosomes; or use the automatic agarose microsphere separation instrument of the group company and its subsidiaries, which is mainly used for glycosylated exosomes The secretion is automatically separated to achieve the purpose of saving manpower. The automatic separation of glycosylated exosomes by agarose microspheres is only an automatic realization of the manual method, and can achieve the same or better than manual separation functions. In this implementation case, manual separation by centrifuge is use...

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Abstract

The present invention relates to a lectin-macromolecule carrier coupling compound for separating a glycosylated exosome in a clinical sample. The lectin-macromolecule carrier coupling compound comprises a macromolecule carrier; and lectin coupled to the outer side of the macromolecular carrier. The lectin-macromolecular carrier coupling compound provided by the invention can be used for simply, conveniently, rapidly and accurately separating the glycosylated exosomes in the clinical sample; the compound is simple in operation, high in separation efficiency and good in repeatability, and the separated exosome is complete in form and free of breakage or fragmentation and can be directly used for glycosylated exosome liquid detection or directly used for immunological related detection or directly used for gene detection and analysis by extracting related nucleic acid in the exosome.

Description

technical field [0001] The invention belongs to the technical fields related to biomedicine and bioseparation, and relates to a lectin-macromolecular carrier coupling complex used for separating glycosylated exosomes in clinical samples. Background technique [0002] Exosomes are biologically active tiny vesicles released when cell multivesicular endosomes fuse with the plasma membrane, with a diameter of about 30-150 nm, and are one of the important mediators of intercellular communication; under healthy human physiological conditions Exosomes can transport DNA, protein, mRNA, miRNA and other biologically active substances between cells, participate in various processes such as cell communication, cell migration, tumor cell growth, etc., and complete the transmission of intercellular substances and information. Literature research has found that almost all types of cells secrete exosomes, and exosomes can be extracted from body fluids such as blood, saliva, urine, cerebrosp...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N1/28G01N1/40B01J20/285B01D15/38B01J20/30
CPCG01N1/28G01N1/40B01D15/3804B01J20/285B01L3/5021B01J20/28019B01J20/28004B01J20/3212B01J20/321B01J20/3274B01J20/3475B01J20/3425B01L2300/0854B01L2200/142B01D21/262B01J20/24B01J20/261B01J20/262B01J20/28016G01N1/34G01N33/54306G01N33/6872
Inventor 林长青陈天圣郝坤黄鹤高琦李艳召许智慧郄霜赵广珍
Owner 北京尧景基因技术有限公司
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