Affinity ligands and methods relating thereto

Abstract

Affinity ligands useful for mild elution affinity chromatography, including affinity ligands specific for immunoglobulins M, A, and E, are disclosed as are method of identifying and using such affinity ligands.

1 - 37 . (canceled) 
     
     
         38 . A method of selecting an affinity ligand that specifically binds to a target molecule under neutral buffer conditions and has reduced binding strength to the target molecular under mild elution conditions, the method comprising the steps of:
 a) expressing a naive affinity ligand library to produce a plurality of affinity ligands;   b) providing a solid support linked to a target;   c) contacting the solid support with the plurality of affinity ligands;   d) washing the solid support with a wash buffer to remove unbound affinity ligands, wherein the wash buffer comprises neutral buffer conditions;   e) contacting the solid support with an elution buffer comprising (i) a pH of about 4.0 to about 5.5 or (ii) about 1-2 M MgCl 2 ; and   f) identifying affinity ligands that substantially dissociate from the solid support in the elution buffer.   
     
     
         39 . The method of  claim 38 , wherein the plurality of affinity ligands is encoded by a plurality of nucleic acid sequences. 
     
     
         40 . The method of  claim 39 , wherein the plurality of nucleic acid sequences comprise a heterologous promoter operably linked thereto. 
     
     
         41 . The method of  claim 38 , wherein the plurality of affinity ligands are expressed on a plurality of phage. 
     
     
         42 . The method of  claim 38 , wherein the elution buffer comprises a pH of about 4.0 to about 5.5 and a relatively low salt concentration. 
     
     
         43 . The method of  claim 38 , wherein the elution buffer comprises about 1 M to 2 M MgCl 2  and a relatively neutral pH. 
     
     
         44 . The method of  claim 38 , wherein the elution buffer comprises about 1 M to 2 M MgCl 2  and a pH of about 6.0 to 8.0. 
     
     
         45 . The method of  claim 38 , wherein the wash buffer comprises a pH of 6.0-8.0. 
     
     
         46 . The method of  claim 38 , wherein the wash buffer comprises a relatively low salt concentration. 
     
     
         47 . The method of  claim 38 , wherein the target is an immunoglobulin. 
     
     
         48 . The method of  claim 38 , wherein the target is an immunoglobulin M (IgM), an immunoglobulin A (IgA), or an immunoglobulin E (IgE). 
     
     
         49 . The method of  claim 38 , wherein the plurality of affinity ligands is a plurality of antibodies or derivatives thereof. 
     
     
         50 . The method of  claim 38 , wherein the plurality of affinity ligands is a plurality of Fab fragments or derivatives thereof. 
     
     
         51 . The method of  claim 38 , wherein the affinity ligand library is not preselected for characteristics favoring reduced binding strength to the target molecule under mild elution conditions. 
     
     
         52 - 58 . (canceled)