Erythrocyte membrane separation medium and erythrocyte membrane separation method

A technology of red blood cell membrane and separation solution, which is applied in the field of preparation of red blood cell membrane, can solve the problems of fluorescence chromatography reaction interference, chromatography detection interference, and the influence of hemoglobin residues, etc., so as to avoid hemoglobin interference and enhance the effect of purity

Active Publication Date: 2020-08-14
TIANJIN DEXIANG BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are three main reasons for the interference: 1) Hemoglobin itself has color, and the membrane sample is fixed on the NC membrane, showing visible color interference; 2) The porphyrin ring of hemoglobin has obvious oxidation reaction activity, which can catalyze horseradish peroxide The substrate of the enzyme (HRP) undergoes a color reaction, which causes interference in ELISA and part of the chromatographic detection in which HRP participates; 3) The porphyrin ring of hemoglobin itself has autofluorescence, and the fluorescence reaction is relatively strong, which interferes with the fluorescence chromatographic reaction
[0005] Existing methods for preparing erythrocyte membranes do not consider the influence of residual hemoglobin on subsequent experiments, nor can they effectively remove residual hemoglobin

Method used

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  • Erythrocyte membrane separation medium and erythrocyte membrane separation method
  • Erythrocyte membrane separation medium and erythrocyte membrane separation method
  • Erythrocyte membrane separation medium and erythrocyte membrane separation method

Examples

Experimental program
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Effect test

Embodiment 1

[0031] Example 1 Measurement method of hemoglobin concentration

[0032] This example illustrates a method for the determination of hemoglobin concentration, which was used to determine the hemoglobin concentration in prepared erythrocyte membrane samples in the Examples below. The concentration of hemoglobin was detected using human hemoglobin (HB) enzyme-linked immunosorbent assay kit.

[0033] Detection principle: Coat the ELISA plate with anti-human HB antibody. During the experiment, the human HB in the sample or standard will be combined with the coating antibody, and the free components will be washed away. Biotinylated anti-human HB antibody and horseradish peroxidase-labeled avidin were added sequentially. The anti-human HB antibody binds to the human HB bound to the coated antibody, and biotin and avidin specifically bind to form an immune complex, and free components are washed away. Add chromogenic substrate (TMB), TMB turns blue under the catalysis of horseradi...

Embodiment 2

[0052] Example 2 Traditional erythrocyte membrane extraction method (comparative example)

[0053] In this example, the red blood cell membrane is prepared by the currently commonly used red blood cell membrane extraction method. The preparation process is as follows:

[0054] 1) Take 3 mL of type A (or other blood group) anticoagulated mixed whole blood, add it to a 15 mL centrifuge tube containing 10 mL of 0.01 mol / L PBS (pH 7.2), centrifuge at 3000 r / min for 5 min, discard the supernatant and the supernatant. White blood cell, platelet layer, obtain about 1.5mL packed red blood cells;

[0055] 2) Wash 3 times with 4°C pre-cooled 0.01mol / L PBS (pH 7.2) equivalent to 3 times the packed red blood cells, and centrifuge at 5000r / min for 15min at 4°C each time;

[0056] 3) Add 0.01mol / L PBS (pH 7.2) pre-cooled at 4°C and mix with the precipitate at the ratio of V:V = 40:1, place at 4°C for 2 h, then centrifuge at 9000r / min for 20min, discard the supernatant ;

[0057] 4) Re...

Embodiment 3

[0062] Example 3 Extraction of erythrocyte membrane using the erythrocyte membrane separation solution provided herein

[0063] In this example, the separation solution provided herein was used to replace the PBS buffer used in the extraction process of erythrocyte membranes in Example 2. In order to compare with the result of Example 2, the operation procedure itself is basically not changed.

[0064] The separation liquid used in this example includes: adenine 0.3 g / L, trehalose 7 g / L, sodium chloride 0.7 g / L, and sodium dihydrogen phosphate 0.02 g / L.

[0065] The specific preparation process of the red blood cell membrane of the present embodiment is as follows:

[0066] 1) Take 3 mL of type A (or other blood type) anticoagulated mixed whole blood, add it to a 15 mL centrifuge tube containing 10 mL of separation medium, centrifuge at 3000 r / min for 5 min, discard the supernatant and the white blood cell and platelet layer under the supernatant, and obtain about 1.5 mL p...

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PUM

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Abstract

The invention provides an erythrocyte membrane separating medium. The erythrocyte membrane separating medium comprises 0.2 to 0.4 g/L of adenine, 6 to 9 g/L of trehalose, 0.5 to 1.0 g/L of sodium chloride and 0.01 to 0.03 g/L of sodium dihydrogen phosphate. The invention further provides a method for preparing an erythrocyte membrane by using the separating medium. The erythrocyte membrane prepared by the method is basically free of hemoglobin residue.

Description

technical field [0001] This paper relates to an erythrocyte membrane separation solution for preparing erythrocyte membranes, and also relates to a method for preparing erythrocyte membranes using the erythrocyte membrane separation solution. Background technique [0002] In biological experiments and clinical tests, it is often necessary to detect or use erythrocyte membrane antigens. These membrane antigens are often difficult to purify from red blood cell membranes, or the purification affects their own biological activities, such as antigenicity. However, in many cases, these assays can be performed directly using erythrocyte membranes (or membrane fragments) with membrane antigens. [0003] The preparation of erythrocyte membrane in the prior art is mainly prepared by the hypotonic method. For example, CN101109755A discloses the process of preparing erythrocyte membrane protein by hypotonic method, including adding pre-cooled 0.01mol / L Tric-HCl and mixing with erythro...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N1/28G01N1/40C12N5/078
CPCC12N5/0641C12N2509/00G01N1/28G01N1/4077G01N2001/4083
Inventor 王丽王秀柱黄志刚王伟权庞伟
Owner TIANJIN DEXIANG BIOTECHNOLOGY CO LTD
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