B-cell cultivation method

1 . A method for co-cultivating a single peripheral blood B-cell for production of an antibody, wherein said B cell is deposited into a well of a multi-well plate using fluorescence activated cell sorting (FACS) comprising the steps of:

a) supplementing a co-cultivation medium with a feeder mix, the feeder mix consisting of additives promoting the activation or survival of B cells or antibody secretion,

wherein the co-cultivation medium supplemented with the feeder mix contains no other additives promoting the activation or survival of B cells or antibody secretion but those of feeder mix; and

b) co-cultivating the B-cell with feeder cells in the presence of the co-cultivation medium supplemented with the feeder mix,

wherein neither of a thymocyte cultivation supernatant, a macrophage cultivation supernatant, or a T-cell cultivation supernatant is added to the co-cultivation medium supplemented with the feeder mix,

wherein the final concentration of the additives promoting the activation or survival of B cells or antibody secretion in the co-cultivation medium supplemented with the feeder mix is:

0.05-0.35 ng/ml phorbol myristate acetate (PMA),

0.02-0.2 ng/ml IL-1beta,

0.02-0.2 ng/ml TNFalpha,

0.5-5 ng/ml IL-2,

0.1-1 ng/ml IL-10,

0.1-1 ng/ml IL-6; and

wherein said B-cell produces an antibody.

2 . The method according to claim 1 , wherein the final concentration of the additives promoting the activation or survival of B cells or antibody secretion in the co-cultivation medium supplemented with the feeder mix is:

0.05-0.35 ng/ml phorbol myristate acetate,

0.2 ng/ml IL-1beta,

0.2 ng/ml TNFalpha,

5 ng/ml IL-2,

1 ng/ml IL-10, and

1 ng/ml IL-6.

3 . The method according to claim 1 , wherein the final concentration of the additives promoting the activation or survival of B cells or antibody secretion in the co-cultivation medium supplemented with the feeder mix is:

0.2-0.35 ng/ml phorbol myristate acetate,

0.064 ng/ml IL-1beta,

0.064 ng/ml TNFalpha,

1.6 ng/ml IL-2,

0.32 ng/ml IL-10, and

0.32 ng/ml IL-6.

4 . The method according to claim 1 , wherein the final concentration of the additives promoting the activation or survival of B cells or antibody secretion in the co-cultivation medium supplemented with the feeder mix is:

0.05-0.2 ng/ml phorbol myristate acetate,

0.064 ng/ml IL-1beta,

0.064 ng/ml TNFalpha,

1.6 ng/ml IL-2,

0.32 ng/ml IL-10, and

0.32 ng/ml IL-6.

5 . The method according to claim 1 , wherein

the IL-1beta has an activity of 5.5-14×10 8 IU/mg,

the TNFalpha has an activity of 2.3-2.9×10 8 U/mg,

the IL-2 has an activity of 6-7×10 6 IU/mg,

the IL-10 has an activity of 6-7.5×10 5 IU/mg, and

the IL-6 has an activity of 9.2-16.1×10 8 U/mg.

6 . The method according to claim 1 , wherein the feeder cells are EL4-B5 cells.

7 . A method for producing an antibody comprising the co-cultivation method according to claim 1 .

8 . The method according to claim 3 , wherein the feeder cells are EL4-B5 cells.

9 . A method for producing an antibody comprising the co-cultivation method according to claim 2 .

10 . The method according to claim 4 , wherein the feeder cells are EL4-B5 cells.

11 . The method according to claim 5 , wherein the feeder cells are EL4-B5 cells.

12 . The method according to claim 1 , where in the co-cultivation medium comprises RPMI 1640 medium supplemented with 10% (v/v) FCS, 1% (w/v) of 200 mM glutamine solution comprising penicillin and streptomycin, 2% (v/v) of 100 mM sodium pyruvate solution, and 1% (v/v) of a 1 M HEPES buffer.

13 . The method according to claim 12 , wherein the co-cultivation medium further comprises 0.05 mM beta-mercaptoethanol.