Application of anti-human CD45RA monoclonal antibody marked by fluorescein isothiocyanate
A fluorescein isothiocyanate, labeling technology, applied in the field of mouse immunoglobulin, biology, can solve problems such as killing
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Embodiment 1
[0021] The ability of 3A4 to recognize antigen and its blocking effect on BD company's anti-CD45RA monoclonal antibody (clone name L48) were detected by flow cytometry inter-standard method ( figure 1 ). Proceed as follows:
[0022] 1. Count KG1a cells and adjust the concentration to 1×10 7 / ml, 100μl cell suspension was taken from each flow tube; 4 reaction tubes were set up in the experiment, which were A. Negative control tube; B. 3A4 antigen recognition ability detection tube; C. Anti-CD45RA monoclonal antibody tube; Anti-CD45RA monoclonal antibody tube.
[0023] 2. Add 50 μl of 3A4 culture supernatant to tubes B and D, and incubate at 4°C for 30 minutes;
[0024] 3. Add PBS and centrifuge at 1000g for 10 minutes to wash away unbound 3A4;
[0025] 4. Add 2 μl GAM-FITC to tube A and tube B respectively, add 2 μl CD45RA-FITC to tube C and tube D respectively;
[0026] 5. Add PBS and centrifuge at 1000g for 10 minutes to wash away unbound antibodies;
[0027] 6. Detecti...
Embodiment 2
[0030] Identification of 3A4 immunoglobulin subclasses: using flow cytometer inter-standard method, KG1a cell line as the reaction cell, 3A4 as the primary antibody, immunoglobulin subclass identification kit as the secondary antibody, and the detection of different immunoglobulins as the secondary antibody Anti-time positive rate and average fluorescence intensity ( figure 2 ). Proceed as follows:
[0031] 1. Count KG1a cells and adjust the concentration to 1×10 7 / ml, take 100μl cell suspension for each flow tube;
[0032] 2. Add 50 μl of 3A4 culture supernatant to each tube, and incubate at 4°C for 30 minutes;
[0033] 3. Add PBS and centrifuge at 1000g for 10 minutes to wash away the bound 3A4;
[0034] 4. Add the secondary antibodies in the immunoglobulin kit, respectively FITC-labeled goat anti-mouse IgG1, IgG2a, IgG2b, IgG3, IgM, Igκ, Igλ, and incubate at 4°C in the dark for 30 minutes;
[0035] 5. Add PBS and centrifuge at 1000g for 10 minutes to wash away unboun...
Embodiment 3
[0039] The anti-human CD45RA mouse immunoglobulin and its heavy chain and light chain variable region genes provided by the present invention are obtained through the following steps:
[0040] 1. The development of ZCH-6-3A4 monoclonal antibody: basically according to the classical mouse-mouse hybridoma method reported by Koller & Milstein, using children's acute undifferentiated lymphocytic leukemia (uALL) cells (the immunophenotype of which is HLA-DR+, CD19+, CD10-, CD33-, TdT+, CD7-, CD41a-) were used as immunogens, and 107 leukemia cells were injected intraperitoneally into 8-week-old female Balb / C mice, once a week, 4 times in total, at the 4th time On the 3rd day after the injection, the mice were killed by dislocation, and the splenocytes were aseptically collected and mixed with the mouse myeloma cell line NS-1 cells in the logarithmic growth phase (ATCC product of the United States) at a ratio of 6:1 and mixed with 50% polyethylene glycol. Diol (PEG, Sigma, USA, molec...
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