Patent ID: 11918635
Assignee: NEOCURA BIO-MEDICAL TECHNOLOGY CO., LTD
Field: Measurement (Instruments)
Classification: CPC G  A  Y | IPC A  G

Claim 2:
3. A method for detecting an immunogenicity of a tumor neoantigen, comprising:
adding human interleukin (IL)-4 into a first Roswell Park Memorial Institute (RPMI) medium comprising 10% (v/v) heat inactivated human serum AB, 100 U/ml penicillin, 100 U/ml streptomycin, 1 mM sodium pyruvate, 0.1 mM non-essential amino acids, 10 mM 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES), 2.5 μg/ml amphotericin B, and 0.05 mM 2-mercaptoethanol to obtain a first culture solution having an IL-4 concentration of 20 ng/ml;
adding granulocyte-macrophage colony-stimulating factor (GM-CSF) into the first culture solution, to obtain a second culture solution having a GM-CSF concentration of 100 ng/ml;
culturing 5×105 thawed human peripheral blood monocytes (PBMCs) in a 24-well plate containing 500 μl of the second culture solution per well and incubating the second culture solution containing PBMCs for 48 h at 37° C.;
adding human IL-7, polyinosinic-polycytidylic acid (Poly I:C), and a first antigenic peptide fragment of a human influenza virus into the second culture solution containing PBMCs to obtain a third culture solution having a human IL-7 concentration of 5 ng/ml and a Poly I:C concentration of 20 μg/ml;
adding a second RPMI medium comprising 10 ng/ml of human IL-7, 10 ng/ml of IL-15, and 40 U/ml of human IL-2 into each well of the 24-well plate to obtain a fourth culture solution containing PBMCs, wherein the total volume of the fourth culture solution containing PBMCs per well is 1 ml;
replacing 500 μl of the fourth culture solution with 500 μl of RPMI medium comprising 10 ng/ml of human IL-7, 10 ng/ml of IL-15, and 40 U/ml of human IL-2;
collecting non-adherent PBMCs from the 24-well plate, washing the non-adherent cells with RPMI medium twice, and then culturing the non-adherent cells in RPMI medium without any cytokines for 48 h, to obtain human PBMC-derived T lymphocytes;
combining 200 μl of RPMI medium comprising 10% (v/v) heat inactivated human serum AB, 100 U/ml penicillin, 100 U/ml streptomycin, 1 mM sodium pyruvate, 0.1 mM non-essential amino acids, 10 mM 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES), 2.5 μg/ml amphotericin B, 0.05 mM 2-mercaptoethanol, 2×105 thawed human PBMCs, 1×105 cells of the human PBMC-derived T lymphocytes, and a second antigenic peptide fragment of a human influenza virus to obtain a fifth culture solution; and
conducting an ELISPOT assay on the fifth culture solution.