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.

What is claimed is: 
     
         1 . A method for cloning a nucleic acid encoding an antibody light chain variable domain or an antibody heavy chain variable domain from an antibody-producing B-cell, the method comprising:
 (a) amplifying the nucleic acid encoding the antibody light chain variable domain or the antibody heavy chain variable domain by polymerase chain reaction (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, and   (b) 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 ligation-independent cloning, wherein the pool of amplified nucleic acids encoding the antibody light chain variable domain or the antibody heavy chain variable domain is inserted into the expression plasmid.   
     
     
         2 . The method of  claim 1 , wherein the method further comprises transforming competent bacteria with the plasmid comprising the nucleic acids encoding the antibody light chain variable domain or the antibody heavy chain variable domain, culturing the transformed bacteria, and isolating the expression plasmid comprising the nucleic acids encoding the antibody light chain variable domain or the antibody heavy chain variable domain from the bacteria. 
     
     
         3 . The method of  claim 1 , wherein the B-cell is a rabbit B cell. 
     
     
         4 . The method of  claim 1 , wherein the B-cell is a single deposited B-cell. 
     
     
         5 . The method of  claim 1 , wherein the B-cell is cultivated for about 7 days prior to cloning the nucleic acid encoding the antibody light chain variable domain or the antibody heavy chain variable domain from the antibody-producing B-cell. 
     
     
         6 . The method of  claim 1 , 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. 
     
     
         7 . A method for producing an antibody, the method comprising: recovering the antibody from the cultivation medium of a eukaryotic cell, wherein the eukaryotic cell comprises nucleic acid encoding the antibody, whereby the nucleic acid encoding the antibody is obtained by
 (a) 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 an 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, and   (b) 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 ligation-independent cloning, wherein the pool of nucleic acids encoding the antibody light chain variable domain or the antibody heavy chain variable domain is inserted into the expression plasmid.   
     
     
         8 . The method of  claim 7 , wherein the B-cell is a rabbit B-cell. 
     
     
         9 . The method of  claim 7 , wherein the B-cell is a single deposited B-cell. 
     
     
         10 . The method of  claim 7 , wherein the B-cell is cultivated for about 7 days prior to amplifying the nucleic acid encoding the antibody light chain variable domain or the antibody heavy chain variable domain from the antibody-producing B-cell. 
     
     
         11 . The method of  claim 7 , wherein the B-cell is a single-deposited B-cell, and further wherein the B-cell produces more than 20 ng/ml of antibody in 7 days of co-cultivation of the B-cell with feeder cells. 
     
     
         12 . The method of  claim 7 , 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. 
     
     
         13 . The method of  claim 7 , wherein method further comprises removing PCR primers after the PCR amplification step. 
     
     
         14 . The method of  claim 7 , wherein the inserting of the amplified nucleic acids encoding the antibody light chain variable domain or the antibody heavy chain variable domain into a eukaryotic expression plasmid is by ligation-independent cloning. 
     
     
         15 . The method of  claim 7 , wherein about 300 ng nucleic acid is used in the insertion step.