Method for generating high affinity antibodies

1. A method for selecting primary antibody-producing cells that express antibodies exhibiting high binding affinity for an antigen of interest, the method comprising:

(a) contacting a population of primary antibody-producing cells that express antibody to an antigen of interest with a low concentration of a monomeric antigen for a time sufficient for the antigen to bind to antibody on the surface of the cells, wherein the low concentration is less than 2 nM;

(b) washing the cells for a period of time to remove unbound antigen; and

(c) collecting cells bound to the antigen, thereby selecting cells expressing high affinity antibodies.

2. The method of claim 1 , wherein the concentration of antigen is between 1 nM and 2 nM.

3. The method of claim 1 , wherein the concentration of antigen is less than 1 nM.

4. The method of claim 1 , wherein cells of the population of cells express antibody that is membrane-bound antibody.

5. The method of claim 1 , wherein the antigen comprises a detectable label.

6. The method of claim 5 , wherein the detectable label is conjugated directly to the antigen.

7. The method of claim 6 , wherein the detectable label is conjugated to antigen via a pair of binding partners.

8. The method of claim 7 , wherein one binding partner of the pair of binding partners is labeled with a detectable label.

9. The method of claim 7 , wherein the pair of binding partners is biotin/streptavidin.

10. The method of claim 5 , wherein the antigen is biotinylated and the biotinylated antigen is contacted with streptavidin comprising the detectable label.

11. The method of claim 10 , wherein cells that bind the biotinylated antigen are contacted with the streptavidin after step b).

12. The method of claim 5 , wherein the detectable label is a fluorescent label.

13. The method of claim 12 , wherein step c) comprises using fluorescence-activated cell sorting to collect cells that bind the antigen.

14. The method of claim 5 , further comprising collecting the cells that bind detectable label to isolate cells that express antibodies of high affinity.

15. The method of claim 1 , wherein the period of time for washing the cells is less than about 60 minutes.

16. The method of claim 1 , wherein said washing the cells for a period of time comprises one (1) wash for about 10 minutes to about 60 minutes total.

17. The method of claim 1 , wherein said washing the cells for a period of time comprises two (2) washes for about 5 minutes to about 30 minutes per wash.

18. The method of claim 1 , wherein the cells collected in step c) are sorted as single cells that express antibodies of high affinity.

19. The method of claim 1 , wherein the primary antibody-producing cells are obtained from spleen, lymph node, peripheral blood and/or bone marrow.

20. The method of claim 1 , wherein the affinity of the antibody is in the range of from about 0.1 pM to about 25 nM (K D ).

21. The method of claim 20 , wherein the affinity of the antibody is from 0.1 nM to 25 nM (K D ).

22. The method of claim 1 , further comprising: isolating antibody-encoding nucleic acid from the cells collected in step (c).

23. The method of claim 22 , further comprising transfecting a host cell with a vector comprising the antibody-encoding nucleic acid; and growing the transfected host cell under conditions to support expression of antibody by the host cell.

24. The method of claim 23 , wherein the vector is an expression vector.

25. The method of claim 24 , wherein the host cell is Chinese hamster ovary (CHO) cell.

26. The method of claim 1 , wherein the cells collected in step c) are sorted into a pool of cells that express antibodies of high affinity.