Monoclonal antibodies directed to peptide in the context of mhc and methods of making and using monoclonal antibodies

Abstract

Phycoerythrin (PE) and peptide:MHCII (p:MHCII) reactive monoclonal antibodies; methods to generate monoclonal antibodies including, for example, peptide:MHC (p:MHC) reactive monoclonal antibodies; compositions including monoclonal antibodies; and uses thereof.

What is claimed is: 
     
         1 . A method comprising:
 immunizing a subject with a composition comprising an antigen, wherein the antigen comprises a peptide-MHC complex (p:MHC);   isolating a population of cells from the subject;   enriching a subpopulation of cells from the population of cells, wherein enriching a subpopulation of cells comprises excluding cells that do not bind to p:MHC, and wherein enriching a subpopulation of cells further comprises at least one of
 increasing the proportion of B cells in the population that bind the antigen, 
 identifying expression of a lymphocyte marker, 
 identifying mononuclear cells, 
 identifying expression of a B cell marker, 
 testing for viability, and 
 testing for antigen specificity; and 
   forming a hybridoma from a cell selected from the subpopulation of cells.   
     
     
         2 . The method of  claim 1 , the method further comprising screening the hybridoma for production of an antibody specific for the p:MHC. 
     
     
         3 . The method of  claim 1  wherein the antigen comprises a monomeric peptide-MHC complex. 
     
     
         4 . The method of  claim 1  wherein the antigen comprises a peptide-MHC Class I complex (p:MHCI) or a peptide-MHC Class II complex (p:MHCII). 
     
     
         5 . The method of  claim 1  wherein the antigen comprises at least two peptide-MHC complexes, wherein the MHC of each peptide-MHC complex comprises the same MHC and wherein the peptide of each peptide-MHC complex is different. 
     
     
         6 . The method of  claim 5  wherein the method comprises immunizing a subject with a composition comprising an antigen comprising one peptide-MHC complex and subsequently immunizing the subject with a composition comprising an antigen comprising another peptide-MHC complex. 
     
     
         7 . The method of  claim 1  wherein enriching the subpopulation of cells comprises enriching a subpopulation of cells capable of binding to a multimeric form of the antigen. 
     
     
         8 . The method of  claim 7  wherein the multimeric form of the antigen comprises at least one of a biotin, a desthiobiotin, and a fluorescent biotin derivative. 
     
     
         9 . The method of  claim 7  wherein a multimeric form of the antigen comprises at least one of a marker and a photosynthetic pigment, and wherein excluding cells that do not bind to p:MHC comprises excluding cells that bind to the marker or the photosynthetic pigment. 
     
     
         10 . The method of  claim 7  wherein enriching cells capable of binding to a multimeric form of the antigen comprises using a magnetic bead, wherein the magnetic bead binds to at least one of a marker and a photosynthetic pigment. 
     
     
         11 . The method of  claim 1  wherein enriching a subpopulation of cells comprises excluding cells that bind to a second peptide-MHC complex, wherein the second peptide-MHC complex comprises the same MHC complex as the p:MHC of the antigen used to immunize the subject, and further wherein the second peptide-MHC complex comprises a different peptide than the p:MHC of the antigen used to immunize the subject. 
     
     
         12 . The method of  claim 1  wherein enriching the subpopulation of the cells comprises using flow cytometric identification and/or sorting. 
     
     
         14 . The method of  claim 1 , wherein excluding cells that do not bind to p:MHC comprises excluding cells that bind to a peptide not bound to an MHC complex and/or to an MHC complex not bound to a peptide. 
     
     
         15 . The method of  claim 1  wherein the subject is a mammal. 
     
     
         16 . The method of  claim 1  wherein the subject is a mouse, a humanized mouse, a rat or a rabbit. 
     
     
         17 . The method of  claim 1  wherein the composition comprising an antigen further comprises an adjuvant. 
     
     
         18 . The method of  claim 17 , wherein excluding cells that do not bind to p:MHC comprises excluding cells that bind to the adjuvant. 
     
     
         19 . A monoclonal antibody, wherein the monoclonal antibody comprises at least one of:
 a heavy chain variable region of FS1, W6, XRI1, or  FIG.  23    and   a light chain variable region of FS1, W6, XRI1, or  FIG.  23   .   
     
     
         20 . A monoclonal antibody, wherein the monoclonal antibody comprises at least one of:
 a heavy chain variable region comprising a complementarity determining region (CDR) of the heavy chain of FS1, W6, XRI1, or  FIG.  23   .   a light chain variable region comprising a complementarity determining region (CDR) of the light chain of FS1, W6, XRI1, or  FIG.  23   .