Immune Repertoire Sequence Amplification Methods and Applications

Abstract

The present invention relates generally to the field of immune binding proteins and method for obtaining immune binding proteins from genomic or other sources. The present invention also relates to nucleic acids encoding the immune binding proteins in which the natural multimeric association of chains is maintained in the nucleic acids and the immune binding proteins made therefrom. For example nucleic acids encoding antibodies that are amplified from a B-cell using the methods of the invention maintain the natural pairing of heavy and light chains from the B-cell. This maintenance of pairing (or multimerization) produces libraries and/or repertoires of immune binding proteins that are enriched for useful binding molecules.

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         21 . A method for making a nucleic acid, comprising the steps of:
 providing a plurality of host cells wherein the host cells comprise a plurality of nucleic acids encoding a plurality of antibody heavy chains and encoding a plurality of antibody light chains, wherein the plurality of host cells express a plurality of different antibodies, and wherein each antibody can bind an antigen;   binding some of the plurality of host cells to a plurality of different antigens wherein each antigen has a unique bar code that identifies the antigen;   isolating individual host cells bound to one of the antigens;   adding a first primer set, a second primer set, a third primer set, a plurality of dNTPs, and a polymerase to the host cells that have bound the antigens,
 wherein the first primer set comprises a forward primer and a reverse primer, wherein one of the primers of the first set has an overlap extension region for a nucleic acid encoding the antibody light chain, the other primer has an overlap extension region for the nucleic acid encoding the bar code, wherein the first primer set amplifies a nucleic acid encoding the antibody heavy chain, 
 wherein the second primer set comprises a forward primer and a reverse primer, wherein one of the primers of the second set has an overlap extension region for the nucleic acid encoding the antibody heavy chain, wherein the second primer set amplifies the nucleic acid encoding the antibody light chain, 
 wherein the third primer set comprises a forward primer and a reverse primer, wherein one of the primers of the second set has an overlap extension region for the nucleic acid encoding the antibody heavy chain, wherein the second primer set amplifies the nucleic acid encoding the bar code; 
   reacting the first primer set, the second primer set, the third primer set, the dNTPs, the polymerase, the nucleic acid encoding the antibody heavy chain, the nucleic acid encoding the antibody light chain, and the bar code whereby a single nucleic acid is obtained that encodes the antibody light chain, the antibody heavy chain, and the bar code.   
     
     
         22 . The method of  claim 21 , wherein the host cell is in an emulsion. 
     
     
         23 . The method of  claim 21 , wherein the host cell is in a well. 
     
     
         24 . The method of  claim 21 , wherein the cell is in a tube. 
     
     
         25 . The method of  claim 21 , wherein the plurality of cells are in an array of a plurality of drops held by surface tension to a surface. 
     
     
         26 . The method of  claim 21 , wherein the plurality of cells are spatially isolated by means of a hydrogel. 
     
     
         27 . The method of  claim 21 , wherein each of the plurality of different antigens is attached to a plurality of beads whereby each different antigen is attached to a different bead. 
     
     
         28 . The method of  claim 27 , wherein each unique barcode for each different antigen is attached to the beads for that antigen. 
     
     
         29 . The method of  claim 21 , wherein the antibody is selected from the group consisting of a scFv, a Fab, a F(ab′)2, a Fab′, a Fv, and a diabody. 
     
     
         30 . The method of  claim 21 , wherein the antibody is selected from the group consisting of an IgG, an IgM, an IgA, an IgD, and an IgE. 
     
     
         31 . The method of  claim 21 , wherein the host cell is selected from the group consisting of a B-cell, a plasma cell, a B memory cell, a pre-B-cell and a progenitor B-cell. 
     
     
         32 . The method of  claim 21 , wherein the plurality of different antigens are from a virus. 
     
     
         33 . The method of  claim 32 , wherein the virus causes an infectious disease. 
     
     
         34 . The method of  claim 33 , wherein the virus is a coronavirus. 
     
     
         35 . The method of  claim 21 , wherein the host cells are obtained from a subject that mounted a protective immune response against a virus. 
     
     
         36 . The method of  claim 35 , wherein the virus is a coronavirus. 
     
     
         37 . The method of  claim 21 , further comprising the step of placing the nucleic acid encoding the antibody light chain, the antibody heavy chain, and the bar code into a vector. 
     
     
         38 . The method of  claim 21 , further comprising the step of sequencing the nucleic acid encoding the antibody light chain, the antibody heavy chain, and the bar code into a vector. 
     
     
         39 . The method of  claim 37 , further comprising the step of sequencing the nucleic acid encoding the antibody light chain, the antibody heavy chain, and the bar code into a vector. 
     
     
         40 . The method of  claim 37 , further comprising the step of placing the vector into a bacterial cell.