Method for producing antibodies using ovine b-cells and uses thereof

Abstract

Herein is reported a cultivation system for cultivating a pool of ovine B-cells or single deposited ovine B-cells in the presence of phorbol myristate acetate (PMA).

1 . A method comprising:
 cultivating ovine B-cells in the presence of phorbol myristate acetate (PMA).   
     
     
         2 . The method of  claim 1 , wherein the cultivating step is performed in the presence of phorbol myristate acetate (PMA), ovine TSN and feeder cells. 
     
     
         3 . The method of  claim 1  further comprising the following steps:
 recovering an antibody from the cultivation supernatant. 
 
     
     
         4 . The method according to  claim 1 , wherein the ovine B-cell is a naive or non-mature ovine B-cell. 
     
     
         5 . The method according to  claim 1 , wherein the B-cell is an IgG positive B-cell (IgG + ). 
     
     
         6 . The method according to  claim 1 , wherein the B-cell is an IgG positive and CD45R positive B-cell (IgG + CD45R + ). 
     
     
         7 . The method according to  claim 1 , wherein the B-cell is an IgG positive and CD21 positive B-cell (IgG + CD21 + ). 
     
     
         8 . A method for producing an antibody, wherein the method comprises the following steps:
 a) cultivating a pool of or a single deposited antibody secreting ovine B-cell in the presence/together with phorbol myristate acetate (PMA),   b) isolating a nucleic acid encoding the amino acid sequence of the variable light chain domain and isolating a nucleic acid encoding the amino acid sequence of the variable heavy chain domain of an antibody, which specifically binds to an antigen,   c) cultivating a cell comprising the nucleic acid isolated in b) or a variant thereof encoding a humanized version of the light and/or heavy chain variable domain within one or more expression cassettes,   d) recovering the antibody from the cell or the cultivation medium and thereby producing an antibody.   
     
     
         9 . A method for producing an antibody, wherein the method comprises the following steps:
 a) providing a population of antibody secreting (mature) B-cells obtained from the blood of a sheep,   b) staining the cells of the population of B-cells with at least one fluorescence dye,   c) depositing single cells of the stained population of B-cells or a pool of cells from the stained population of B-cells in individual containers,   d) cultivating the deposited individual B-cells in the presence of phorbol myristate acetate (PMA),   e) determining the binding specificity of the antibodies secreted in the cultivation of the individual or pool of B-cells,   f) determining the amino acid sequence of the variable light and heavy chain domain of specifically binding antibodies by a reverse transcriptase PCR and nucleotide sequencing, and thereby obtaining a monoclonal antibody variable light and heavy chain domain encoding nucleic acid,   g) introducing the monoclonal antibody light and heavy chain variable domain encoding nucleic acid in an expression cassette for the expression of an antibody,   h) introducing the nucleic acid in a cell,   i) cultivating the cell and recovering the antibody from the cell or the cell culture supernatant and thereby producing an antibody.