A Lectin-Magnetic Carrier Conjugated Complex for the Isolation of Glycosylated Exosomes from Clinical Samples

A magnetic carrier and exosome technology, applied in carrier binding/immobilization of peptides, analytical materials, biological testing, etc., can solve the problems of large steric hindrance, rupture of exosome vesicles, low separation efficiency, etc., reducing space The effect of steric hindrance, reducing steric hindrance and improving separation efficiency

Active Publication Date: 2022-03-25
北京尧景基因技术有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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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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  • A Lectin-Magnetic Carrier Conjugated Complex for the Isolation of Glycosylated Exosomes from Clinical Samples
  • A Lectin-Magnetic Carrier Conjugated Complex for the Isolation of Glycosylated Exosomes from Clinical Samples
  • A Lectin-Magnetic Carrier Conjugated Complex for the Isolation of Glycosylated Exosomes from Clinical Samples

Examples

Experimental program
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Effect test

Embodiment example 2

[0069] Example 2 Preparation of Glycosylated Exosome Separation Composition

[0070] 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.

[0071] 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.

[0072] 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

[0074] Example 3 Method of using glycosylated exosome isolation composition

[0075] 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".

[0076] The detailed experimental steps of the present invention include:

[0077] 1. Preparation before the experiment

[0078] 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 ...

Embodiment example 4

[0087] This example mainly further illustrates the separated samples of glycosylated exosomes. The samples to be separated that can be used in the present invention are mainly: serum, plasma, saliva, tissue or cell culture supernatant, urine, cerebrospinal fluid, lymph fluid any one; for conventional 1.5ml centrifuge tube, add the pretreated sample volume 50-500ul of the serum, plasma, saliva, cerebrospinal fluid, lymph, tissue or cell culture supernatant, urine, preferably 50 -300ul, more preferably 100-200ul; in the sample pretreatment process, the samples of serum, plasma, saliva, cerebrospinal fluid, and lymph are only centrifuged, so for 50-500ul, 50-300ul can be selected, and 100 can be selected. -200ul of the sample can be pre-treated to obtain the required volume of pre-treated samples; and the tissue or cell culture supernatant, urine samples in the sample pre-treatment process, in addition to the centrifugation step, also need Concentrate 10-1000 times, so in order t...

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Abstract

The present invention relates to a lectin-magnetic carrier coupling complex for separating glycosylated exosomes in clinical samples, the lectin-magnetic carrier coupling complex comprising: a magnetic carrier; Lectin on the outside of the carrier. The lectin-magnetic carrier conjugate complex provided by the present invention can quickly, accurately and automatically separate glycosylated exosomes in clinical samples, with high separation efficiency, and the separated exosomes are complete in shape without rupture or fragmentation, It can be directly used for the detection of glycosylated exosome liquid, or directly for immunological related detection, or directly extract relevant nucleic acids in exosomes for nucleotide sequence detection and analysis.

Description

technical field [0001] 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 [0002] 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 in cell communication, cell migration, tumor cell growth and other processes, and complete the transfer of intercellular substances and information. Literature studies have found that almost all types of cells secrete exosomes, and exosomes can be extracted from body fluids such as blood, saliva, urine, cereb...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K17/10C07K17/08G01N33/68G01N33/569
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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