Solubilization of antigen components for removal from tissues

Abstract

The present invention relates to methods for removing antigens from tissues by sequentially destabilizing and/or depolymerizing cytoskeletal components and removing and/or reducing water-soluble antigens and lipid-soluble antigens. The invention further relates to tissue scaffolding and decellularized extracellular matrix produced by such methods.

1 - 20 . (canceled) 
     
     
         21 . A kit comprising (i) a solution for solubilizing water soluble antigens and (ii) a solution for solubilizing lipid soluble antigens. 
     
     
         22 . The kit of  claim 21 , wherein the solution for solubilizing water soluble antigens comprises a buffering agent, a reducing agent, a protease inhibitor, and one or more salts suitable for maintaining protein solubility. 
     
     
         23 . The kit of  claim 22 , wherein the buffering agent maintains a pH of the solution for solubilizing water soluble antigens in the range of about 4 to about 11. 
     
     
         24 . The kit of  claim 22 , wherein the buffering agent maintains a pH of the solution for solubilizing water soluble antigens of at least about 8. 
     
     
         25 . The kit of  claim 22 , wherein the one or more salts are a monovalent or a divalent anion. 
     
     
         26 . The kit of  claim 22 , wherein the one or more salts are a metal halide salt. 
     
     
         27 . The kit of  claim 26 , wherein the metal halide salt is selected from the group consisting of LiF, LiCl, LiBr, LiI, NaF, NaCl, NaBr, NaI, KF, KCl, KBr, KI, RbF, RbCl, RbBr, RbI, CsF, CsCl, CsBr, CsI, BeF 2 , BeCl 2 , BeBr 2 , BeI 2 , MgF 2 , MgCl 2 , MgBr 2 , MgI 2 , CaF 2 , CaCl 2 , CaBr 2 , CaI 2 , SrF 2 , SrCl 2 , SrBr 2 , SrI 2 , BaF 2 , BaCl 2 , BaBr 2 , BaI 2 , and mixtures thereof. 
     
     
         28 . The kit of  claim 22 , wherein the reducing agent is selected from the group consisting of Tributylphosphine (TBP), beta mercaptoethanol, 2-mercaptoethylamine, tris(2-carboxyethyl)phosphine (TCEP), cysteine-HCl, and dithiothreitol (DTT). 
     
     
         29 . The kit of  claim 22 , wherein the solution for solubilizing water soluble antigens comprises one or more of an antibacterial agent or an antifungal agent. 
     
     
         30 . The kit of  claim 21 , wherein the solution for solubilizing water soluble antigens does not comprise an amphiphile. 
     
     
         31 . The kit of  claim 21 , wherein the solution for solubilizing water soluble antigens does not comprise a detergent. 
     
     
         32 . The kit of  claim 21 , wherein the solution for solubilizing water soluble antigens comprises a non-detergent sulfobetaine. 
     
     
         33 . The kit of  claim 21 , wherein the solution for solubilizing water soluble antigens comprises Tris-HCl, dithiothreitol (DTT), a protease inhibitor, and KCl. 
     
     
         34 . The kit of  claim 21 , wherein the solution for solubilizing lipid soluble antigens comprises a buffering agent for maintaining a pH of the solution for solubilizing lipid soluble antigens, a reducing agent, a protease inhibitor, one or more salts suitable for maintaining protein solubility and an amphiphile. 
     
     
         35 . The kit of  claim 34 , wherein:
 (a) the amphiphile is a zwitterionic detergent; or   (b) the buffering agent maintains the pH of the solution for solubilizing lipid soluble antigens in the range of about 4 to about 11; or   (c) the buffering agent maintains the pH of the solution for solubilizing lipid soluble antigens of at least about 8.0.   
     
     
         36 . The kit of  claim 34 , wherein the amphiphile is a sulfobetaine. 
     
     
         37 . The kit of  claim 36 , wherein the sulfobetaine is selected from the group consisting of 3-[N,N-Dimethyl(3-myristoylaminopropyl)ammonio]propanesulfonate (amidosulfobetaine-14; ASB-14); amidosulfobetaine-16 (ASB-16); 4-n-Octylbenzoylamido-propyl-dimethylammonio sulfobetaine (ASB-C80); 3-(N,NDimethyloctylammonio) propanesulfonate inner salt (SB3-8); N-Decyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (SB 3-10), N-Decyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (SB 3-12), N-Tetradecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (SB 3-14); 3-(N,N-Dimethylpalmitylammonio) propanesulfonate (SB3-16); 3-(N,N-Dimethyloctadecylammonio) propanesulfonate (SB3-18); 3-(1-Pyridinio)-1-propanesulfonate (NDSB-201); 3-(Benzyldimethylammonio) propanesulfonate (NDSB-256); NDSB-211, NDSB-195, NDSB-221; 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), 3-[(3-Cholamidopropyl) dimethylammonio]-2-hydroxy-1-propanesulfonate (CHAPSO), and mixtures thereof. 
     
     
         38 . The kit of  claim 21 , wherein the solution for solubilizing lipid-soluble antigens comprises Tris-HCl, dithiothreitol (DTT), a protease inhibitor, KCl, and ASB-14. 
     
     
         39 . A kit for removing immunogenic antigens from a tissue, comprising:
 (a) a solubilizing water-soluble antigen solution comprising a first buffering agent, a first reducing agent, a first protease inhibitor, and one or more salts suitable for maintaining protein solubility; and   (b) a solubilizing lipid-soluble antigen solution comprising a second buffering agent, a second reducing agent, a second protease inhibitor, one or more salts suitable for maintaining protein solubility and an amphiphile.   
     
     
         40 . The kit of  claim 39 , wherein:
 (a) the water-soluble antigen solution comprises Tris-HCl, dithiothreitol (DTT), the first protease inhibitor, and KCl; and/or   (b) the lipid-soluble antigen solution comprises Tris-HCl, dithiothreitol (DTT), the second protease inhibitor, KCl, and ASB-14; and/or   (c) the water-soluble antigen solution is substantially free of an amphiphile; and/or   (d) the amphiphile in the solubilizing lipid-soluble antigen solution comprises a non-detergent sulfobetaine; or   (e) the kit further comprises instructions for removing immunogenic antigens from a tissue; or   (f) the kit further comprises instructions for generating a tissue scaffold depleted of antigens comprising an extracellular matrix (ECM) structure and an ECM tensile strength that is substantially the same as the ECM prior to antigen removal.