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Method for separating exosome in biological sample, kit and application thereof

A technology of biological samples and kits, applied in the field of medical testing, can solve the problems of ineffective evaluation of clinical effects, increased cost and operational complexity, and low accuracy of results, achieving high accuracy, good integrity, and high yield and The effect of improving the purity

Pending Publication Date: 2022-05-10
JIANGSU MICRODIAG BIOMEDICINE TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are some defects and deficiencies: 1) It is greatly affected by individual differences in urine, so the accuracy of the results is low, and the detection rate is low and the retest rate is high; 2) The joint detection of multiple RNA targets increases the cost and operational complexity ; 3) Only for the distinction between prostate cancer and non-cancer, there is no effective evaluation of the clinical effect on the distinction between normal and prostatitis / hyperplasia / cancer

Method used

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  • Method for separating exosome in biological sample, kit and application thereof
  • Method for separating exosome in biological sample, kit and application thereof
  • Method for separating exosome in biological sample, kit and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0095] Example 1 Enrichment and purification method of total urine exosomes

[0096] 1.1 Enrichment of total exosomes in urine-anion exchange chromatography column enrichment of total exosomes

[0097] The steps of enriching total exosomes by anion exchange chromatography column are as follows:

[0098] Add 8-20ml equilibration buffer to the anion exchange column to wash the column to achieve equilibrium;

[0099] Pour off the effluent, add urine samples (multiple batches), discard the effluent;

[0100] Add 20-50ml of washing solution to wash the column protein and impurities, and discard the effluent;

[0101] Then use 5mL eluent to elute the combined substance, and collect the effluent;

[0102] Then 2 mL of eluent was used to elute the bound substances, and the effluent was collected, which was the total exosomes in urine.

[0103] Control group 1: Enrichment of total exosomes by PEG precipitation method

[0104] Mix urine with PEG20000 precipitant at a ratio of 1:1; ...

Embodiment 2

[0116] The equilibrium buffer optimization of embodiment 2 ion exchange enrichment

[0117] The following four kinds of buffer systems shown in table 1 were compared respectively: in the anion exchange column equilibrium process, system 1, system 2 and system 3 were selected conventional phosphate buffer saline or Tris-HCl buffer; system 4 was in conventional buffer ( Tris-HCl buffer, PBS buffer or phosphate buffer) are added with high-valent cations to bind the anions released from the ion-exchange column, so that the ion-exchange column generates more positive charges for binding to exosomes.

[0118] Table 1

[0119]

[0120] 1) Sample pretreatment: 2000ml of total urine was mixed, collected in a sterile container, centrifuged at 14000g at 4°C for 10min and filtered with 0.22um to remove cells and cell debris. Then 100ml / tube, divided into 15 parts.

[0121] 2) Anion-exchange chromatography column was used to enrich total exosomes respectively, and each system in Table...

Embodiment 3

[0163] Example 3 Optimization of Elution Conditions for Ion Exchange Enrichment

[0164] The enrichment conditions of urine exosomes were optimized by using ion-exchange chromatography to achieve a simple operation process and better enrichment effect. The effects of different fractions and eluent concentrations shown in Table 8 were compared respectively. The equilibration buffer used in this embodiment is PBS buffer 0.02mol / L (pH 7.4). The washing solution was 100 mM sodium chloride buffer and 50 mM sodium phosphate buffer.

[0165] Table 8

[0166]

[0167] The specific steps are:

[0168] 1) Sample pretreatment: Mix 2000ml of total urine, collect in a sterile container, centrifuge at 14000g / centrifuge for 10min at 4°C, and filter with 0.22um to remove cells and cell debris. Then 100ml / tube, divided into 15 parts.

[0169] 2) The ion exchange chromatography method (the method is the same as in Example 2) is used to enrich the total exosomes in urine, and the elution...

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Abstract

The invention discloses a kit for separating exosomes in a biological sample, which is characterized by comprising an equilibrium buffer solution, a washing solution and an eluent, the equilibrium buffer solution contains a basic buffer solution and high-valence cations, and the high-valence cations are selected from any one or more of Ca < 2 + >, Mg < 2 + >, Fe < 3 + >, Fe < 2 + >, Al < 3 + >, Mn < 2 + >, Cu < 2 + > and Zn < 2 + >. The invention also discloses a method for separating the exosome by using the kit for separating the exosome in the biological sample. The invention further discloses application of the kit for separating the exosome in the biological sample to exosome collection in diagnosis or distinguishing of prostate diseases, postoperative evaluation of prostate cancer and prognosis evaluation of prostate cancer, and application of the kit in preparation of therapeutic drugs taking the exosome as a functional component. The invention further discloses a kit for diagnosing the prostate diseases and a kit for typing the prostate diseases.

Description

technical field [0001] The invention relates to the technical field of medical detection, in particular to a method for separating exosomes in biological samples, a kit and applications thereof. Background technique [0002] The prostate is the largest gland among the accessory glands of the male reproductive system and plays an important role in both the urinary and reproductive systems. Prostate disease mainly includes prostatitis, benign prostatic hyperplasia and prostate cancer. It is a common disease of adult male genitourinary system, seriously affects the health status and quality of life of men, and has become one of the new serious public health problems. [0003] At present, the pathogenesis and pathophysiological changes of prostate diseases are not very clear. There are still no clear standards to follow in many aspects such as clinical diagnosis, treatment methods and curative effect evaluation. Rectal ultrasonography (TRUS), prostate-specific antigen (PSA), d...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071C12N5/078C12Q1/6883C12Q1/6886G01N15/02G01N21/76A61K35/14A61K35/16A61K35/22A61K47/46
CPCC12N5/0684C12N5/0634C12Q1/6886C12Q1/6883G01N21/76G01N15/02A61K35/22A61K35/14A61K35/16A61K47/46C12N2509/00G01N2800/342C12Q2600/158C12Q2600/118C12Q2600/112
Inventor 刘云飞陈雅奇董肇楠渠香云王弢
Owner JIANGSU MICRODIAG BIOMEDICINE TECH CO LTD
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