Freeze-dried NK cell surface antigen quality control material and preparation method thereof

A technology of NK cells and surface antigens, applied in the field of immunological detection, can solve the problems that reference substances cannot be used in different equipment or detection platforms, storage and transportation conditions are harsh, and data cannot be compared, etc., and achieve good antigen expression stability. and cell characteristics reproducibility, saving transportation and storage costs

Inactive Publication Date: 2018-01-12
巴德生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These reference substances have been widely used, but there are the following problems: the storage and transportation conditions are harsh, requiring low temperature (2-8°C) or ultra-low temperature (-80°C); the storage period is short, and the shelf life of whole blood is 3 months; The process is complicated, for example, freezing cells in liquid nitrogen requires cell recovery first
The same reference product cannot be used in different equipment or detection platforms, and the data between different equipment cannot be compared at one time

Method used

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  • Freeze-dried NK cell surface antigen quality control material and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] The application of the present invention provides a method for preparing a freeze-dried NK cell surface antigen quality control product, as follows:

[0032] 1. Collect blood through veins, collect human peripheral blood cells, spread the cell suspension on the sucrose solution, and centrifuge to make cells of different densities aggregate, and collect human peripheral lymphocytes;

[0033] 2. Rinse with phosphate buffered saline containing serum;

[0034] 3. Add anti-human CD3 and anti-human CD16, CD56 antibodies, protect from light, incubate at 15°C for 30 minutes, collect cells after centrifugation, re-suspend cells in phosphate buffer, elute unlabeled excess antibodies, and harvest antibodies by centrifugation labeled cells;

[0035] 4. To harvest the cells, follow 10 3 Cells / mL concentration, add fixative, mix thoroughly by shaking, and fix at 5°C for 36 hours in the dark;

[0036] 5. Elute the fixative, use a flow cytometer to count the number of positive cells...

Embodiment 2

[0041] The application of the present invention provides a method for preparing a freeze-dried human NK lymphocyte quality control product, as follows:

[0042] 1. After washing the buffy coat (Buffy coat), collect human peripheral blood cells, spread the cell suspension on commercial human lymphocyte separation medium, and centrifuge to make cells of different densities aggregate, and collect human peripheral lymphocytes;

[0043] 2. Rinse with Hank's buffer;

[0044] 3. Add anti-human CD3 and anti-human CD16, CD56 antibodies, protect from light, incubate at 30°C for 15 minutes, collect cells after centrifugation, resuspend cells in Hank's solution, elute unlabeled excess antibodies, and harvest antibody-labeled cells by centrifugation cell;

[0045] 4. To harvest the cells, follow 10 8 Cells / ml concentration, add fixative, mix thoroughly by pipetting, and fix at 30°C for 1 hour in the dark;

[0046] 5. Elute the fixative, use a flow cytometer, and count the number of posi...

experiment example 1

[0051] Two different batches of cryopreserved labeled human peripheral CD3- / CD16+ / CD56+ cells each took 3 different samples and performed 20 tests. The counts of CD3-negative and CD16, CD56-positive cells per microliter are shown in the table below. Human peripheral T lymphocytes that show cryointervention labeling can maintain good consistency between different operators and laboratories, and the results are shown in the following table:

[0052] Table 1

[0053]

[0054] The results show:

[0055] 1. The cells prepared by this method maintain complete cell morphology and cell contents, and are similar or identical to fresh cells;

[0056] 2. The cells prepared by this method maintain the expression of surface antigens, and there is no loss of target antigens;

[0057] 3. The positive cells prepared by this method have the same detection results on different flow cytometers, which can form a reference and control between different models;

[0058] 4. The number of positiv...

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Abstract

The invention aims to provide a freeze-dried NK cell surface antigen quality control material and a preparation method thereof. The reference substance is subjected to surface or intracellular specific antibody labeling by peripheral lymphocytes and can be applied to direct detection of flow cytometry. In the preparation process of the reference substance, the concentration of specific cells is calibrated after positive cell counting of the specific antigen, so the reference substance can provide reference for relative counting detection of the flow cytometry and can also serve as a referencesubstance or a counting reference in absolute counting detection, particularly applied to reference and calibration of flow cytometry calculated by a volumetric method at different time, different equipment and different personnel.

Description

technical field [0001] The application of the present invention relates to a freeze-dried NK cell surface antigen quality control product and a preparation method thereof, belonging to the technical field of immunological detection. Background technique [0002] Leukocyte differentiation antigen is a cell surface marker that appears or disappears during the normal differentiation and maturation of leukocytes (including platelets, vascular endothelial cells, etc.) in different lineages, different stages and activation processes. Most of them are transmembrane proteins or glycoproteins, including extracellular region, transmembrane region and cytoplasmic region. Cluster of differentiation (cluster of differentiation, CD) T cells in the process of differentiation and maturation, different developmental stages and different subtypes of lymphocytes can express different differentiation antigens, which is an important symbol to distinguish lymphocytes. [0003] Flow cytometry is ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/533G01N15/14C12N5/0783
Inventor 李博
Owner 巴德生物科技有限公司
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