Preparation of red blood cells with a modified level of blood group antigen expression and their use in the quality control of blood typing reagents
A technology for red blood cells and antigens, applied in the field of cells with altered blood group antigen expression levels, and can solve problems such as inability to detect
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Embodiment 1
[0133] Decreased Expression Level of Antigen B Acted by α-Galactosidase
[0134] 10 units of α-galactosidase extracted from green coffee beans were purchased from Glyko (Cat. No. X-5001).
[0135] 100 microliters of erythrocytes of blood type AB were washed three times with phosphate buffered saline (PBS).
[0136] Control response (ctrl):
[0137] Fifty microliters of encapsulated erythrocytes were added to 40 microliters of 100 mM citrate buffer pH 6.5 and 22.5 microliters of 500 mM citrate buffer pH 6.5.
[0138] Enzymatic reaction (enz):
[0139] Add 50 µl of encapsulated cells to 40 µl of 4 units of α-galactosidase in 100 mM pH 6.5 citrate buffer and 22.5 µl of 500 mM pH 6.5 citrate buffer in the liquid.
[0140] The mixture was reacted in a 37°C water bath with occasional mixing. The end times of the reactions were 6 hours and 9 hours, respectively, and the method of stopping was to remove the equivalent of 15 microliters of packed red blood cells, and wash the ce...
Embodiment 2
[0148] Decreased Expression Level of Antigen A Acted by α-N-acetylgalactosaminidase
[0149] α-N-acetylgalactosaminidase (Cat. No. X-5001) was purchased from Glyco.
[0150] 100 microliters of erythrocytes of blood type AB were washed three times with phosphate buffered saline (PBS).
[0151] Control response (ctrl):
[0152]6 microliters of encapsulated erythrocytes were added to 100 microliters of 100 mM citrate buffer pH 4 and 26.5 microliters of 500 mM citrate buffer pH 6.5.
[0153] Enzymatic reaction (enz):
[0154] Add 6 µl of encapsulated cells to 100 µl of 100 milliunits (mU) of α-N-acetylgalactosaminidase in 100 mM pH 4 citrate buffer and 26.5 µl of 500 mM pH 6.5 in citrate buffer.
[0155] The mixture was reacted in a 37°C water bath with occasional mixing. The end time of the reaction is 6 hours, 12 hours and 24 hours respectively, and the method of stopping is to take out 2 microliters of wrapped red blood cells and add them to 100 microliters of CelStab re...
Embodiment 3
[0167] Process for the preparation of alpha-galactosidase from coffee beans.
[0168] α-Galactosidase was extracted from green coffee beans (Coffea mesocarpus) according to the method of Courtois and Petek. (Courtois, J.E. and Petek, J., α-Galactosidase from Coffee Beans, (1996) Methods of Enzymology, 8:565-571).
[0169] The extracted enzyme was concentrated to a centrifugal ultrafiltration device (Millipore) and dialyzed against citrate phosphate buffer (100 mM, pH 6.0). The activity of the crude enzyme extract does not need to be determined. The total protein concentration was 96 mg / ml.
[0170] Enzymatic approach to reduce expression of B antigen
[0171] Control response (ctrl):
[0172] Washed and wrapped red blood cells (300 μl) of blood type AB were added to Eppendorf centrifuge tubes filled with citrate phosphate buffer (500 μl, 100 mM, pH 6.0 and 200 μl, 500 mM, pH 6.0) middle.
[0173] Enzymatic reaction (enz):
[0174] Washed and wrapped red blood cells ...
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