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Method for extracting neutrophil azurophilic granules from human blood transfusion waste

A neutrophil and waste technology, applied in the field of medical clinical testing, can solve problems such as being only suitable for scientific research, difficult to popularize in industrialization, and not suitable for medical clinical research and in vitro diagnosis of diseases.

Inactive Publication Date: 2018-10-16
武汉博百欧生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] In 2005, Medical Theory and Practice, Vol.18(3), 272-2274, Isolation and Purification of Neutrophils, authors Li Peng and Xing Jie mentioned a commonly used human neutrophil isolation method, such as Ficoll-Hypaque density gradient centrifugation and Percoll density gradient centrifugation, but because the density of neutrophils is similar to that of red blood cells, this method has the disadvantage of being difficult to completely separate neutrophils from red blood cells
[0009] To sum up, there are currently many methods for isolating human and animal neutrophils shown and reported in domestic and foreign literature, but the raw materials used in these methods are all from normal human or animal whole blood. Scarce resources, their circulation and use are subject to strict control, so the method of extracting neutrophil azurophilic granules from human blood as a raw material has great difficulties in industrialization and popularization
At the same time, due to the non-biological homology of animal proteases and human proteases, the method of extracting neutrophil azurophilic granules from animal blood as a raw material is only applicable to the scientific research stage and is not suitable for human medicine after industrialization. Clinical research and in vitro diagnosis of diseases

Method used

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  • Method for extracting neutrophil azurophilic granules from human blood transfusion waste
  • Method for extracting neutrophil azurophilic granules from human blood transfusion waste
  • Method for extracting neutrophil azurophilic granules from human blood transfusion waste

Examples

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Embodiment 1

[0034] In this embodiment, a total of 630 white blood cell filters recovered from the central blood bank were used. After the blood cell mixture is obtained by primary elution, the red blood cells in the mixture are removed by lysing the red blood cells, and the leukocyte enrichment precipitate is obtained by centrifugation and washing, and then the leukocyte lysate obtained by cracking the leukocyte enrichment precipitate through a nitrogen booster device is finally added to it. Density gradient separation of the bioparticle solvent followed by centrifugation to obtain 33.2 g of neutrophil azurophilic granule tissue precipitate. The specific implementation steps are as follows:

[0035] 1. The raw material for obtaining neutrophil azurophilic granules comes from human blood transfusion leukocyte filter. This filter belongs to the discarded medical supplies after human blood transfusion. The human blood leukocyte filter is required to be stored in an environment of 4°C-8°C u...

Embodiment 2

[0046] In this embodiment, the composition of the azurophilic granulosa cell tissue precipitate layer extracted in Example 1 is analyzed and verified, and the verification process is as follows: grind and dissolve 0.1 g of the azurophilic granule cell tissue precipitate layer separated and extracted in Example 1, and After the grinding is uniform, the analysis and verification of the components of the cell grinding solution is carried out. The verification method is polyacrylamide gel electrophoresis (SDS-PAGE), stained with Coomassie blue, and then analyzed with ChemiDoc MP imager of BioRad to analyze the results of electrophoresis gel. Test results such as figure 1 As shown, according to the molecular weight of proteinase 3 (PR3) (29-33kD), the molecular weight of myeloperoxidase (MPO) (59kD) and the molecular weight of bacterial permeability factor (BPI) (55kD), it can be obtained from figure 1 The results of the protein glue deduced that the grinding solution of neutrophi...

Embodiment 3

[0048] This example further qualitatively verifies the results obtained from the protein gel analysis in Example 2. The verification method is protein glue transfer and Western Blotting. The primary antibodies used are rabbit polyclonal anti-MPO antibody (1:1000, Jackson) and rabbit polyclonal anti-PR3 antibody (1:300, Jackson). Test results such as figure 2 shown, from figure 2 It can be clearly seen that the grinding solution of neutrophil azurophilic granules contains at least the two protein antigens of MPO and PR3, which proves that it can be used as the starting protein solution raw material for the purification of MPO and PR3 antigens.

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Abstract

The invention provides a method for extracting neutrophil azurophilic granules from human blood transfusion waste. The method comprises the following steps: flushing a blood transfusion leukocyte filter to obtain leukocyte; then removing erythrocyte by utilizing erythrocyte lysate and lysing leukocyte in nitrogen booster equipment by using leukocyte lysate; in addition, adding a density gradient separation biogranule solvent to carry out centrifugal separation on cell tissues to obtain target cell tissues. By adopting the method, the neutrophil azurophilic granules used as purification raw materials of protein antigens such as protease 3 (PR3), myeloperoxidase (MPO) and bacterial permeability factors (BPI) can be industrially separated and extracted, which is of great significance in medical clinical examination.

Description

technical field [0001] The invention relates to the technical field of medical clinical testing, in particular to a method for extracting neutrophil azurophilic granules from human blood transfusion waste. Background technique [0002] Neutrophils account for 50-70% of white blood cells and are the most abundant white blood cells in the peripheral blood circulation and immune system. Neutrophils play a very important role in the body's nonspecific immune defense response, and are important phagocytic cells for the body to resist microbial pathogens, especially the invasion of suppurative bacteria. The cytoplasm of neutrophils is light pink and contains many fine granules, among which the light purple ones are Azurophilic Granules and the light red ones are Special Granules. Azurophilic particles account for about 20% of the total number of particles. Under the electron microscope, the particles are relatively large, with a diameter of 0.6-0.7µ; m, round or oval, and have a ...

Claims

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Application Information

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IPC IPC(8): C12N9/64
CPCC12N9/6421
Inventor 孙鹏康雅明
Owner 武汉博百欧生物科技有限公司
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