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Freeze-drying protection liquid, freeze-drying method and application of erythrocyte membrane fragment

A technology of freeze-dried protective solution and red blood cell membrane, which is applied to freeze-dried cell membrane fragments and its application in the detection of blood type reversal. Many other problems to achieve the effect that is conducive to standardization

Inactive Publication Date: 2021-06-25
TIANJIN DEXIANG BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The use of freshly prepared blood group antigens makes the process complicated, and too much manpower is consumed in the preparation of antigens
The short-term storage method of membrane fragments has many disadvantages, such as the storage period is less than one week, and as the storage period increases, the antigenicity decreases. It is not suitable for mass production of red blood cell membrane fragments as raw materials, resulting in serious batch-to-batch differences.

Method used

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  • Freeze-drying protection liquid, freeze-drying method and application of erythrocyte membrane fragment
  • Freeze-drying protection liquid, freeze-drying method and application of erythrocyte membrane fragment
  • Freeze-drying protection liquid, freeze-drying method and application of erythrocyte membrane fragment

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1 Preparation of erythrocyte membrane fragments

[0039] The red blood cell debris preparation process is as follows:

[0040] 1) Take 3 mL of anticoagulated mixed whole blood (taken from 6-10 blood samples of type A (or other blood types), add it to a 15 mL centrifuge tube filled with 10 mL of 0.01mol / L PBS (pH7.2), and Centrifuge at 3000r / min for 5min, discard the supernatant and the white blood cell and platelet layer under the supernatant, and obtain about 1.5mL packed red blood cells;

[0041] 2) Wash 3 times with 4°C pre-cooled 0.01 mol / L PBS (pH7.2) equivalent to 3 times the volume of packed red blood cells, and centrifuge at 5000 r / min for 15 minutes at 4°C each time;

[0042] 3) Add 4°C pre-cooled 0.01 mol / L PBS (pH7.2) at a ratio of V:V = 40:1 and mix with the sediment, place at 4°C for 2 hours, then centrifuge at 9000r / min for 20 minutes, discard clear;

[0043] 4) Repeat step 3) 4 more times until no red blood cells are visible to the naked eye, a...

Embodiment 2

[0045] Example 2 Preparation of Lyoprotectant Solution

[0046] Using water for injection, prepare 3 kinds of lyoprotectant solutions according to the following components and concentrations:

[0047] 1#: 20mM glucose, 10mM lactose, 140mM trehalose, 39mM NaCl, 5.3mM KCl, 0.125mM KH 2 PO 4 , 1.2mM Na 2 HPO 4 ;

[0048] 2#: 10mM glucose, 12mM lactose, 141mM trehalose, 31mM NaCl, 4.3mM KCl, 0.115mM KH 2 PO 4 , 1.1mM Na 2 HPO 4 ;

[0049] 3#: 15mM glucose, 10mM lactose, 131mM trehalose, 35mM NaCl, 5.0mM KCl, 0.115mM KH 2 PO 4 , 1.1mM Na 2 HPO 4 .

[0050] In the description below, the corresponding lyoprotectant solutions are represented by corresponding numbers (1#, 2# or 3#).

Embodiment 3

[0051] Example 3 Lyophilization of erythrocyte membrane fragments

[0052] The fresh erythrocyte membrane fragments prepared in Example 1 and the lyoprotectants prepared in Example 2 were mixed according to a volume ratio of 1:9, and added to a vial with a volume of 5 mL, so that the liquid content of each vial was 1 mL. Put the vial in a refrigerator at 4 degrees Celsius for 30 minutes, then drop it to -70 degrees Celsius at a rate of 10 degrees Celsius / minute, and keep it at this temperature for more than 2 hours to freeze the freeze-dried product. Then start to vacuumize and dry once, so that the partition temperature and pressure of the lyophilizer are kept at about -50 degrees Celsius and 2-4 Pa respectively, and the time is 18 hours. Finally, secondary drying is performed, during which the temperature of the partition is set at 20 degrees Celsius, the pressure is about 2-4 Pa, and the duration is 10 hours to obtain freeze-dried erythrocyte membrane fragments.

[0053] F...

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Abstract

The invention provides a freeze-drying protection liquid of an erythrocyte membrane fragment. The protection solution is prepared from 10-20 mM of glucose, 6-12 mM of lactose, 121-141 mM of trehalose, 29-39 mM of NaCl, 4.3-5.3 mM of KCl, 0.115-0.125 mM of KH2PO4 and 0.6-1.2 mM of Na2HPO4. The invention also provides a method for preparing a freeze-dried erythrocyte membrane fragment by using the freeze-drying protection liquid. The erythrocyte membrane fragment freeze-drying protection liquid and the freeze-drying method provided by the invention can be used for preparing the freeze-dried erythrocyte membrane fragment, and the freeze-dried erythrocyte membrane fragment can be used for blood type detection and are beneficial to standardization of a reverse typing process.

Description

technical field [0001] The invention relates to a freeze-dry protection solution for red blood cell membrane fragments and a freeze-drying method for using the freeze-dry protection solution to freeze-dry red blood cell membrane fragments. The invention also relates to the freeze-dried cell membrane fragment prepared by the freeze-drying method and its application in blood type reverse typing detection. Background technique [0002] At present, microcolumn gel blood group cards are mainly used for clinical blood type detection, and its essence is to use agglutination technology to detect antigen-antibody reactions. Among them, the positive typing test is to use the blood group monoclonal antibody reagent to detect the blood group antigen on the red blood cell membrane, and the reverse typing test is to use the red blood cell reagent to detect the corresponding antibody in the serum. The erythrocyte reagent uses a erythrocyte preservation solution to preserve erythrocytes. ...

Claims

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Application Information

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IPC IPC(8): A01N1/00G01N33/80
CPCA01N1/00G01N33/80
Inventor 庞伟王丽王秀柱黄志刚王伟权
Owner TIANJIN DEXIANG BIOTECHNOLOGY CO LTD
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