Anti-human Tim3 monoclonal antibody and application thereof
An antibody and sequence technology, applied in the fields of biotechnology and medicine, can solve the problems of small Tim3 antibody rejection and high antibody affinity
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Embodiment 1
[0121] Embodiment 1, preparation of anti-human Tim3 antibody
[0122] 1. Construction of anti-human Tim3 monoclonal antibody hybridoma cell line
[0123] Three Balb / c mice and three C57b1 / 6 mice aged 4-6 weeks were selected, and human Tim3 (hTim3) was used as the antigen, and 100 μg ricin was subcutaneously immunized in the groin, once every two weeks, and a total of 3 mice were immunized. Second-rate. Blood was collected from the tail vein after the third immunization, and the antibody production was detected by indirect ELISA. The appropriate immunized mice were selected, the eyes were removed and the neck was removed, and the splenocytes of the mice were aseptically removed, and the cells were fused according to conventional methods to prepare anti-human mice. Tim3 monoclonal antibody hybridoma cell line.
[0124] Anti-human Tim3 monoclonal antibody hybridoma cells were screened by ELISA. ELISA plates were coated with 10 μg / ml human Tim3 (hTim3), overnight at 4°C and blo...
Embodiment 2
[0133] Example 2, Anti-human Tim3 antibody affinity identification
[0134] 1) Detection of Tim3 antibody affinity by ELISA
[0135] The HKT8 that adopts the hybridoma cell strain of embodiment 1 to obtain carries out following experiment:
[0136] Reagents used include: human Tim3 (hTim3, as antigen), and 96-well microtiter plate (Nunc company), plating buffer, washing solution, blocking solution, horseradish peroxidase-labeled avidin, chromogenic substrate and stop solution.
[0137] Wherein, the plating buffer is a phosphate buffer with a pH of 7.0;
[0138] The washing solution is a solution obtained by adding Tween 20 to a phosphate buffer solution with a pH of 7.0, and the content of Tween 20 in the washing solution is 0.05% by volume.
[0139] The blocking solution is a solution obtained by adding BSA to the washing solution, and the content of BSA in the blocking solution is 10 g / L.
[0140] Horseradish peroxidase-labeled avidin (that is, horseradish peroxidase-lab...
Embodiment 3
[0164] Embodiment 3, FACS detection anti-human Tim3 antibody specificity
[0165] The HKT8 that adopts the hybridoma cell strain of embodiment 1 to obtain carries out following experiment:
[0166] The tested cells used in the experiment were CHO-K1 cell line (CHO-hTim3) highly expressing human Tim3 and CHO-K1 cell line (CHO-cyTim3) highly expressing monkey Tim3. FACS analysis of cells as image 3 .
[0167] Cultivate the cells to be tested to 80% fullness with a T75 bottle, wash once with PBS, digest with trypsin, and collect the cells (the number of cells in each bottle is 10 6 cells), add RPMI1640 medium by pipetting, centrifuge at 1000rpm for 5min, discard the supernatant, and collect the cells. The collected cells were resuspended and washed with about 1 ml of the FACS buffer of Example 2, centrifuged at 1000 rpm for 5 min, the supernatant was discarded, and the precipitate was collected. Then resuspend the cells with 750 μl of FACS buffer to obtain a cell suspension,...
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