HLA antibody specificity detecting method, cell dish and reagent kit
A detection method and specific technology are applied in the field of HLA antibody specific detection method and cell disk and kit, which can solve the problems of increasing difficulty in matching kidney sources, difficulty in finding out HLA antibody specificity, and high PRA value in the serum to be tested. To achieve the effect of good specificity and low cost
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Embodiment 1
[0082] Example 1. Adjustment of HLA Antigen Frequency for Selected Donor Cell Catalogs
[0083] Table 3 HLA antigen adjustment scheme of the present invention
[0084] Active adjustment of antigen types
Frequency of HLA antigens in Chinese population (%)
Adjusted HLA antigen frequency in the cell plate (%)
w4
66
50-55
w6
62
50-55
A1
21
10-15
A2
54
25-30
A3
22
15-19
A11
33
5-15
A24
33
10-20
C7
44
25-30
w9
30
20-25
[0085] Antigen frequency adjustment principle:
[0086] In addition to the HLA antigen frequencies listed in Table 3, the selected donor cell HLA antigens also need to cover all the antigens listed in Table 1, and each antigen appears at least twice.
Embodiment 2
[0087] Embodiment 2 makes cell dish
[0088] Step 1. Select a cell donor:
[0089] Among normal healthy adults, volunteers are randomly selected to donate peripheral blood, and a total of 150 to 250 volunteers are required as the source of candidate cell donors.
[0090] Cell donor HLA tissue type identification, numbering and recording of cell HLA antigen type.
[0091] According to the HLA antigen matching of the donors, the data in Table 3 in Example 1, and the antigen frequency adjustment principle, 96 donors were selected, and 100 ml of venous peripheral blood were extracted respectively.
[0092] Step 2. Using Ficoll-Hypaque to obtain a mononuclear cell layer (PBMC), which contains a large number of peripheral blood lymphocytes. The cells were washed twice with phosphate buffered saline (PBS), and the cells were counted. (please adjust the format)
[0093] Step 3. Ninety-six donor peripheral blood lymphocytes were added to wells of a 96-well cell culture plate, with ...
Embodiment 3
[0104] Example 3. Detection of HLA antibody specificity
[0105] 1. Thaw two cell discs prepared in Example 2 at room temperature (22° C.), one cell disc is used as a base value detection disc, and the other cell disc is used as a sample detection disc. When testing multiple sera at one time, only one base test plate is needed.
[0106] 2. Wash the plate once with RPMI-1640 solution containing 10% FCS, and then wash the plate once with flow cytometer washing buffer.
[0107] 3. Add 25 μl of positive control serum to one well of the base value test plate and the sample test plate.
[0108] 4. Add 25 μl of negative control serum to the remaining 95 wells of the base value detection plate.
[0109] 5. Add 25 μl of negative control serum to another well of the specimen testing plate, and add 25 μl of patient serum to be tested into the remaining 94 wells.
[0110] 6. Incubate all base value detection plates and sample detection plates at 4°C for 30 minutes.
[0111] 7. Wash th...
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