Freeze-dried human lymphocyte CD4 surface antigen quality control material and preparation method thereof

A lymphocyte and surface antigen technology, applied in the field of immunological detection, can solve the problems of harsh storage and transportation conditions, the microspheres easily exceed the collection range, and the storage period is short, saving transportation and storage costs, and improving the stability of antigen expression. and reproducibility of cell properties

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: 1. The storage and transportation conditions are harsh, requiring low temperature (2-8°C) or ultra-low temperature (-80°C); 2. The storage period is short, and the shelf life of whole blood is 3 3. The use process is complicated, for example, liquid nitrogen cryopreservation requires cell recovery first
The same reference product cannot be used on different equipment or testing platforms. Different equipment is not compatible at one time. The equipment needs to be used in conjunction with reagents. The data between different equipment cannot be compared.
[0005] At present, the clinical significance of absolute cell counting in flow cytometry detection is becoming more and more important. At present, the commonly used methods for absolute cell counting are: (1) microspheres. The problem of using counting microspheres for absolute cell counting is that the reagents are expensive; Due to excessive voltage adjustment, the microspheres exceed the data collection range, resulting in detection failure, so the operator's skills are required to be high
Problems in this method: it is necessary to use counting microspheres for proofreading every time, and the price of microsphere reagents is relatively high. , the microspheres are easy to exceed the collection range

Method used

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  • Freeze-dried human lymphocyte CD4 surface antigen quality control material and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] The application of the present invention provides a method for preparing a freeze-dried human lymphocyte CD4 surface antigen quality control product, as follows:

[0033] 1. Collect human peripheral blood cells through venous blood collection, spread the cell suspension on the sucrose solution, and centrifuge to make cells of different densities aggregate, and collect human peripheral lymphocytes;

[0034] 2. Rinse with phosphate buffered saline containing serum;

[0035] 3. Add anti-human CD4 antibody, protect from light, incubate at 18°C ​​for 20 minutes, collect cells after centrifugation, resuspend cells in phosphate buffer, elute unbound excess antibody, and harvest antibody-labeled cells by centrifugation;

[0036] 4. To harvest the cells, follow 10 3 Cells / mL concentration, add fixative, mix thoroughly by shaking, and fix at 6°C for 0.5 hours in the dark;

[0037] 5. Elute the fixative, use a flow cytometer, and count the number of positive cells. According to ...

Embodiment 2

[0042] The application of the present invention provides a method for preparing a freeze-dried human lymphocyte CD4 surface antigen quality control product, as follows:

[0043] 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;

[0044] 2. Rinse with Hank's buffer;

[0045] 3. Add anti-human CD4 antibody, protect from light, incubate at 30°C for 30 minutes, and collect cells after centrifugation. Re-suspend cells in Hank's solution, elute unbound excess antibody, and harvest antibody-labeled cells by centrifugation;

[0046] 4. Follow 10 3 Cells / mL concentration, add fixative solution, mix thoroughly by pipetting, and fix at 25°C for 36 hours in the dark;

[0047] 5. Elute the fixative, use a flow cytometer, and count the number of positive cells. According to t...

experiment example 1

[0052] Take samples from two different batches, 3 bottles from each batch. Add 1ml of ultrapure water to each bottle and place it at 15-30°C for 5-10 minutes to fully absorb and redissolve the freeze-dried cells. After fully mixing, each bottle of cell suspension was evenly distributed to 20-22 flow-type sample loading tubes in the amount of 50 μl / tube, and 150 μl of phosphate buffer was added to each sample loading tube. Detection was performed on BDFACSCalibur and BC Cytomics FC 500 flow cytometers, respectively. The results are shown in the table below:

[0053] Table 1

[0054]

[0055] The results show:

[0056] 1. The cells prepared by this method maintain complete cell morphology and cell contents, and are similar or identical to fresh cells;

[0057] 2. The cells prepared by this method maintain the expression of surface antigens, and there is no loss of target antigens;

[0058] 3. The positive cells prepared by this method have the same detection results on d...

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Abstract

The invention aims to provide a freeze-dried human lymphocyte CD4 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. Moreover, in the preparation process of the reference substance, the concentrationof 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 reference substance 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 human lymphocyte CD4 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] The c...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/533G01N15/14C07K14/73C12N5/0781C12N5/0783
Inventor 李博
Owner 巴德生物科技有限公司
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