Method for preparing immunoglobulin libraries
The invention relates to the generation of immunoglobulin libraries and the identification and production of immunoglobulins having a specific functionality of interest.
1. A method for generating an immunoglobulin library comprising:
isolating RNA from a subset of B cells consisting essentially of CD24+/CD27+/IgM+ cells, and
preparing an immunoglobulin library from the isolated RNA.
2. The method according to claim 1 , further comprising deriving the subset of B cells from a single individual before isolating the subset of B cells.
3. The method according to claim 1 , further comprising displaying immunoglobulins of the subset of B cells on the surface of replicable genetic packages.
4. The method according to claim 1 , wherein the prepared immunoglobulin library is selected from the group consisting of an antibody library, a single chain Fv library and a Fab library.
5. A method for generating an immunoglobulin library, wherein the method comprises the steps of:
a) isolating a subset of B cells from an individual, wherein the subset of B cells consists essentially of CD24+/CD27+/IgM+ cells,
b) isolating RNA from the subset of B cells, c) converting the isolated RNA into cDNA,
d) amplifying immunoglobulin nucleic acid molecules of said cDNA,
e) inserting the amplified immunoglobulin nucleic acid molecules into at least one vector, and
f) transforming at least one host cell with the at least one vector containing the amplified nucleic acid molecules to obtain an immunoglobulin library.
6. The method according to claim 1 , further comprising:
screening the prepared immunoglobulin library for an immunoglobulin having a functionality of interest.
7. The method according to claim 6 , wherein screening the prepared immunoglobulin library for an immunoglobulin having a functionality of interest comprises:
a) contacting the prepared immunoglobulin library with an antigen under conditions conducive to binding,
b) separating and recovering immunoglobulins that bind to the antigen from immunoglobulins that do not bind,
c) isolating at least one recovered immunoglobulin,
d) screening if the immunoglobulin isolated has a functionality of interest, and
e) isolating an immunoglobulin having the functionality of interest.
8. The method according to claim 3 , wherein the replicable genetic package is selected from the group consisting of a phage particle, a bacterium, a yeast, a fungus, a spore of a microorganism, and a ribosome.
9. The method according to claim 8 , wherein the immunoglobulin library is selected from the group consisting of an antibody library, a single chain Fv library, and a Fab library.
10. The method according to claim 2 , further comprising displaying immunoglobulins of the subset of B cells on the surface of a replicable genetic package, wherein the replicable genetic package is selected from the group consisting of a phage particle, a bacterium, a yeast, a fungus, a spore of a microorganism, and a ribosome.
11. The method according to claim 10 , wherein the immunoglobulin library is selected from the group consisting of an antibody library, a single chain Fv library, and a Fab library.
12. The method according to claim 2 , wherein the single individual is a human.
13. The method according to claim 12 , further comprising displaying immunoglobulins of the subset of B cells on the surface of a replicable genetic package, wherein the replicable genetic package is selected from the group consisting of a phage particle, a bacterium, a yeast, a fungus, a spore of a microorganism, and a ribosome.
14. The method according to claim 13 , wherein the immunoglobulin library is selected from the group consisting of an antibody library, a single chain Fv library, and a Fab library.
15. The method according to claim 1 wherein at least 90% of the cells used to prepare the library are CD24+/CD27+/IgM+ B cells.
16. The method according to claim 1 wherein at least 92% of the cells used to prepare the library are CD24+/CD27+/IgM+ B cells.
17. The method according to claim 1 wherein at least 95% of the cells used to prepare the library are CD24+/CD27+/IgM+ B cells.
18. The method according to claim 1 wherein at least 97% of the cells used to prepare the library are CD24+/CD27+/IgM+ B cells.
19. The method according to claim 5 , wherein at least 90% of the cells used to prepare the library are CD24+/CD27+/IgM+ B cells.
20. The method according to claim 5 , wherein at least 92% of the cells used to prepare the library are CD24+/CD27+/IgM+ B cells.
21. The method according to claim 5 , wherein at least 95% of the cells used to prepare the library are CD24+/CD27+/IgM+ B cells.
22. The method according to claim 5 , wherein at least 97% of the cells used to prepare the library are CD24+/CD27+/IgM+ B cells.