Rapid method for cloning and expression of cognate antibody variable region gene segments

Abstract

In the method as reported herein the isolation of nucleic acid segments encoding antibody variable domains and the insertion of the isolated nucleic acid segments in eukaryotic expression plasmids is performed without the intermediate isolation and analysis of clonal intermediate plasmids. Thus, in the method as reported herein the intermediate cloning, isolation and analysis of intermediate plasmids is not required, e.g. by analysis of isolated transformed E. coli cells.

1 - 15 . (canceled) 
     
     
         16 . A method for producing an antibody, the method comprising:
 (i) obtaining an antibody-secreting B-cell from an animal, wherein the B-cell comprises nucleic acid encoding the antibody;   (ii) culturing the B-cell as a single-deposited B-cell, wherein the B-cell and its progeny produce greater than 20 ng/ml of the antibody in 7 days of co-cultivation of the B-cell with feeder cells;   (iii) amplifying the nucleic acid encoding the antibody light chain variable domain or the antibody heavy chain variable domain by PCR using single-stranded cDNA obtained from the RNA of the antibody-secreting B-cell as template, thereby obtaining a pool of nucleic acids encoding the antibody light chain variable domain or the antibody heavy chain variable domain;   (iv) generating single strand extensions of the pool of nucleic acids in the absence of nucleotides using T4 DNA polymerase; and   (v) inserting the amplified nucleic acids encoding the antibody light chain variable domain or the antibody heavy chain variable domain into a eukaryotic expression plasmid by sequence- and ligation-independent cloning, wherein the pool of nucleic acids encoding the antibody light chain variable domain and the antibody heavy chain variable domain, respectively, is inserted into the expression plasmid.   
     
     
         17 . The method of  claim 16 , wherein the B-cell is a rabbit B-cell. 
     
     
         18 . The method of  claim 16  or  claim 17 , wherein the feeder cells are EL4-B5 cells. 
     
     
         19 . The method of  claim 16  or  claim 17 , wherein PCR primers comprising the nucleic acid sequences of SEQ ID NOs:5 and 6 or the nucleic acid sequences of SEQ ID NOs:7 or 8 are used for the amplification of nucleic acid encoding the antibody light chain variable domain or the antibody heavy chain variable domain, respectively. 
     
     
         20 . The method of  claim 16  or  claim 17 , wherein method further comprises removing PCR primers after the PCR amplification step. 
     
     
         21 . The method of  claim 16  or  claim 17 , wherein about 300 ng nucleic acid is used in the insertion reaction.