Application of lectin-magnetic carrier coupling compound in separation of glycosylated exosome

A magnetic carrier and lectin technology, applied in the direction of carrier binding/immobilization of peptides, solid adsorbent liquid separation, separation methods, etc., can solve the problems of large steric hindrance, rupture of exosome vesicles, and low separation efficiency, and achieve The effect of reducing steric hindrance, reducing steric hindrance, and improving separation efficiency

Pending Publication Date: 2022-07-29
北京尧景基因技术有限公司
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  • 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 eluent used in the immunomagnetic bead method is an acidic eluent, which can easily cause exosome vesicle rupture and incomplete morphology. Therefore, it is impossible to accurately separate glycosylated exosomes and apply them Detection, monitoring and diagnosis during the occurrence and development of diseases

Method used

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  • Application of lectin-magnetic carrier coupling compound in separation of glycosylated exosome
  • Application of lectin-magnetic carrier coupling compound in separation of glycosylated exosome
  • Application of lectin-magnetic carrier coupling compound in separation of glycosylated exosome

Examples

Experimental program
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Effect test

Embodiment example 1

[0054] Example 1 Preparation of lectin-magnetic carrier conjugated complex

[0055] The lectin-magnetic carrier conjugated complex includes: a magnetic carrier; and a lectin coupled to the outside of the magnetic carrier, which is prepared by coupling the lectin and the magnetic carrier under specific conditions, and is mainly used for separating clinical samples The glycosylated exosomes obtained after isolation are complete in morphology. in:

[0056] Lectins are mainly used: pineapple agglutinin (Jacalin), peanut agglutinin (PNA), pea agglutinin (VVA and / or VVL), concanavalina agglutinin (ConA), lentil agglutinin (LCA), wheat germin (WGA), soybean lectin (SBA), kidney bean lectin (PVL), snail lectin (HAA and / or HPA), or any combination of two or more lectins, the main reasons are: The combination of different lectins can realize the separation of various types of glycosylated exosomes, such as: LCA, AAL for the separation of fucosylated exosomes; ConA, PVL, SBA, WGA, AAL ...

Embodiment example 2

[0070] Example 2 Preparation of Glycosylated Exosome Separation Composition

[0071] The present invention provides a composition for separating glycosylated exosomes, which specifically includes: a lectin-magnetic carrier conjugated complex, which is used for washing and removing non-specifically bound and unglycosylated exosomes during the separation process. and other impurities, and / or eluate for elution of glycosylated exosomes specifically bound to lectin-magnetic carrier conjugated complexes, individually packaged and present in a set or in the kit.

[0072] In this example, the above-mentioned lectin-magnetic carrier conjugated complex is the LCA-agarose magnetic bead solution with a volume ratio of 50% obtained in Example 1.

[0073] The above cleaning solution is a metal-salt ion-free cleaning buffer or purified water, and can optionally be a metal-salt-free cleaning buffer, such as: the main component includes 10-200mM metal-salt-free TRIS-HCl buffer with a pH of 7...

Embodiment example 3

[0075] Example 3 Method of using glycosylated exosome isolation composition

[0076] The present invention also provides a method for separating glycosylated exosomes, which mainly includes the main experimental steps of using the composition for separating glycosylated exosomes in Example 2. Please refer to the schematic diagram of the separation principle for details. figure 1 "Schematic diagram of the separation of glycosylated exosomes by lectin-magnetic carrier conjugated complexes".

[0077] The detailed experimental steps of the present invention include:

[0078] 1. Preparation before the experiment

[0079] Self-provided equipment or equipment: magnetic stand for manual separation of glycosylated exosomes; or use the fully automatic agarose magnetic bead separation instrument of the group company and its subsidiaries, mainly for automatic glycosylated exosome separation separation, to achieve the purpose of saving manpower. The automatic separation of glycosylated ...

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Abstract

The invention relates to a divisional application with the application number of 202010060055.7. The invention relates to a lectin-magnetic carrier coupling compound for separating glycosylated exosomes in a clinical sample, and the lectin-magnetic carrier coupling compound comprises a magnetic carrier, a binding agent, a binding agent and a binding agent, and lectin coupled to the outside of the magnetic carrier. The lectin-magnetic carrier coupling compound provided by the invention can be used for rapidly, accurately and automatically separating the glycosylated exosome in a clinical sample, the separation efficiency is high, and the separated exosome is complete in form and free from rupture or fragmentation, and can be directly used for glycosylated exosome liquid detection or directly used for related immunology detection, so that the detection efficiency is high. Or directly extracting related nucleic acid in the exosome for nucleotide sequence detection and analysis.

Description

[0001] The application date of the present invention is January 19, 2020, the application number is 202010060055.7, and the invention-creation name is "a lectin-magnetic carrier conjugated complex for separating glycosylated exosomes in clinical samples". A divisional application for a Chinese application. technical field [0002] The invention belongs to the technical field related to biomedicine and biological separation, and relates to a lectin-magnetic carrier conjugated complex for separating glycosylated exosomes in clinical samples, and a separation method. Background technique [0003] Exosomes are tiny biologically active vesicles released when 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 ...

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
CPCG01N1/28G01N1/40B01D15/3804B01J20/285B01J20/28009B01J20/3212B01J20/3274B01J20/28019B01J20/28004B01J20/3425B01J20/3475C07K14/42C07K17/14C07K17/08C07K17/10G01N1/405G01N33/54333
Inventor 林长青陈天圣郝昆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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