Isolation of membrane bound ligand-specific complexes

Abstract

A method to recover or “deracinate” receptors from intact membranes in their native configuration without removing microenvironmental components associated with them is described. The membranes containing the receptors are first associated with ligand-coupled solid supports to achieve association of the membranes through a receptor/ligand complex which includes the microenvironment to the solid support and then removing the membrane from the receptor and its microenvironment by shear forces. The thus isolated receptors and their microenvironments can then be analyzed for their role in cellular differentiation, apoptosis, transformation and the like.

1 . A method to isolate and characterize a membrane-bound receptor along with its microenvironment which method comprises 
 (a) providing a solid support coupled to a ligand which specifically binds said receptor;    (b) treating said solid support with a sample comprising nucleated cells or organelles thereof comprising said membrane-bound receptors, and which cells or organelles have not been surface-treated,    wherein a complex is formed between said membrane-bound receptor and the ligand thus generating a ligand/receptor complex comprising the receptor and its microenvironment, which complex is coupled to solid support through the ligand;    (c) separating the solid support from the remainder of the sample;    (d) subjecting the separated solid support to a force sufficient to dissociate the receptor and its microenvironment from the membrane but insufficient to disrupt the ligand/receptor complex;    thus obtaining solid support coupled to a ligand/receptor complex whereby the receptor retains its microenvironment but is separated from the membrane, and, optionally,    (e) analyzing the microenvironment of the receptor.    
     
     
         2 . The method of  claim 1  wherein step (e) is performed.  
     
     
         3 . The method of  claim 1  which further comprises removing the ligand/receptor complex comprising the receptor and its microenvironment from the solid support.  
     
     
         4 . The method of  claim 1  which further comprises removing the receptor and its microenvironment without ligand from the solid support.  
     
     
         5 . The method of  claim 1  wherein, in step (d), the cells remain intact.  
     
     
         6 . The method of  claim 1  wherein the cells are vertebrate cells.  
     
     
         7 . The method of  claim 6  wherein the cells are tumor cells or diseased cells.  
     
     
         8 . The method of  claim 6  wherein the cells are hematopoietic cells, or cells from adipose, areolar, connective, elastic, epithelial, endothelial, neural, mucous or reticular tissues.  
     
     
         9 . The method of  claim 1  wherein the ligand is an antibody or an immunospecific portion thereof.  
     
     
         10 . The method of  claim 1  wherein the receptor comprises an HLA antigen.  
     
     
         11 . The method of  claim 1  wherein the receptor comprises a tumor associated antigen.  
     
     
         12 . The method of  claim 1  wherein the receptor is a cytokine receptor, a hormone receptor, an opioid receptor, or a steroid receptor.  
     
     
         13 . The method of  claim 1  wherein the force is achieved through extrusion.  
     
     
         14 . The method of  claim 1  wherein the force is achieved through vortexing or shaking.  
     
     
         15 . The method of  claim 1  wherein the force is achieved through sonication.  
     
     
         16 . The method of  claim 1  wherein the solid support comprises beads.  
     
     
         17 . The method of  claim 14  wherein said beads are polyacrylamide beads, polystyrene beads, Sephadex beads, or latex beads.  
     
     
         18 . The method of  claim 1  wherein the solid support is a multi-well plate.  
     
     
         19 . The method of  claim 3  wherein the ligand is coupled to the solid support through a linker containing a photocleavable portion and said removing is effected by exposing the linker to light.  
     
     
         20 . The method of  claim 3  wherein the ligand is coupled to solid support through a linker containing a portion cleavable by an enzyme and said removing is effected by exposing said linker to said enzyme.  
     
     
         21 . The method of  claim 4  wherein said removing is achieved by treating the complexed solid support with a substance that binds said ligand in competition with the receptor.  
     
     
         22 . A method to recover a multiplicity of receptors along with their microenvironments which method comprises: 
 (a) providing a multiplicity of solid support portions each coupled to a different ligand;    (b) treating a sample comprising nucleated cells that comprise at least two cell surface receptors, wherein said cells are not surface treated with said multiplicity of solid support portions under conditions wherein ligand/receptor complexes are formed between said ligands and receptors at said cell surfaces;    (c) removing the solid support portions from the sample; and    (d) subjecting the solid support portions to forces sufficient to remove the receptors and their microenvironments from the surface of said cells but insufficient to disrupt the ligand/receptor complexes, and    (e) analyzing the respective microenvironments of at least two receptors.    
     
     
         23 . The method of  claim 22  which further comprises removing the ligand/receptor complexes or the receptors and their microenvironments from the solid support.  
     
     
         24 . The method of  claim 22  which further includes identifying the receptors.  
     
     
         25 . The method of  claim 23  which further includes identifying the receptors.  
     
     
         26 . The method of  claim 24  identifying components in said micro-environment.  
     
     
         27 . The method of  claim 25  identifying components in said micro-environment.  
     
     
         28 . The method of  claim 24  which further includes organizing the identified receptors into a profile characteristic of the membrane sample.  
     
     
         29 . The method of  claim 25  which further includes organizing the identified receptors into a profile characteristic of the membrane sample.  
     
     
         30 . The method of  claim 22  wherein each of said multiplicity of portion is coupled to a different ligand.  
     
     
         31 . The method of  claim 30  wherein said ligands are monoclonal antibodies.