Depletion of plasma proteins

Abstract

This invention relates to methods of analysis, and in particular to methods for the preliminary fractionation of samples in which low abundance molecules of interest, for example proteins, polysaccharides or fatty acids, are present together with more abundant molecules of little or no interest. In particular, the invention relates to methods of depletion of high abundance proteins from biological samples. Products and kits for use in the method are also disclosed, and form part of the invention. In one aspect, the invention provides a method of depleting a high-abundance molecule from a biological sample, comprising the steps of a) subjecting the sample to affinity depletion using an affinity support with high affinity for a high abundance molecule, and/or b) immunodepletion using an affinity support coupled to an antibody directed against whole or previously fractionated plasma or serum.

1 . A method of depleting a high-abundance molecule from a biological sample, comprising the steps of:
 a) subjecting the sample to affinity depletion using an affinity support with high affinity for the high abundance molecule; and   b) immunodepleting using an affinity support coupled to an avian antibody directed against the high-abundance molecule.   
     
     
         2 . A method according to  claim 1 , in which the antibody is a chicken antibody. 
     
     
         3 . A method according to  claim 2 , in which the antibody is chicken IgY. 
     
     
         4 . A method according to  claim 1 , in which the avian antibody used in step (b) is a first generation polyclonal antibody raised against whole serum or plasma, or against any fraction thereof. 
     
     
         5 . A method according to  claim 1 , in which the avian antibody used in step (b) is a second or higher generation polyclonal antibody raised against plasma or serum which has already been subjected to at least one round of affinity depletion and immunodepletion with an affinity support coupled to IgY directed against homologous plasma or serum. 
     
     
         6 . A method according to  claim 1 , in which step (a) is performed before step (b). 
     
     
         7 . A method according to  claim 1 , in which the high abundance molecule is a protein. 
     
     
         8 . A method according to  claim 7 , in which the protein is albumin or immunoglobulin. 
     
     
         9 . A method according to  claim 1 , in which the biological sample is a biological fluid. 
     
     
         10 . A method according to  claim 1 , in which the biological sample is conditioned medium from a cell or tissue culture, or is a tissue or cell extract. 
     
     
         11 . A method according to  claim 1 , in which the affinity support used in step (a) is a dye affinity chromatography resin. 
     
     
         12 . A method according to  claim 11 , in which the dye is a chlorotriazine compound. 
     
     
         13 . A method according to  claim 12 , in which the affinity support is a Cibacron blue F3GA affinity support. 
     
     
         14 . A method according to  claim 1 , in which the affinity support used in step (a) is a magnetic bead and the magnetic separation is effected by magnetic means. 
     
     
         15 . A method of separation or analysis of a low abundance molecule in a biological sample, comprising the step of depleting at least one high abundance molecule from the sample by a method according to  claim 1 , prior to subjecting the sample to one or more separation or analytical steps for the separation or analysis of the low abundance molecule. 
     
     
         16 . A method of identifying the expression of a low abundance molecule in a mammal, comprising the step of depleting at least one high abundance molecule from a biological sample from the mammal by a method according to  claim 1 , prior to subjecting the sample one or more analytical steps to detect the expression of the low abundance molecule. 
     
     
         17 . A method according to  claim 16 , detects which a change in the expression of the low abundance molecule. 
     
     
         18 . A composition for immunodepletion of a high abundance molecule from a biological sample, comprising an avian antibody preparation directed against the high abundance molecule, coupled to an affinity support. 
     
     
         19 . A composition according to  claim 18 , in which the avian antibody is a chicken antibody. 
     
     
         20 . A composition according to  claim 18 , in which the avian antibody is chicken IgY. 
     
     
         21 . A composition according to  claim 18 , in which the avian antibody is a first generation polyclonal antibody. 
     
     
         22 . A composition according to  claim 21 , in which the antibody is a second or higher generation polyclonal antibody. 
     
     
         23 . A composition according to  claim 18 , in which the high abundance molecule is one present in serum or plasma. 
     
     
         24 . A composition according to  claim 23 , in which the high abundance molecule is albumin or immunoglobulin. 
     
     
         25 . A device for the rapid processing of biological samples, comprising a chamber having two openings, in which
 a) each opening is adapted to fit sealingly to a receptacle,   b) the sample can be transferred from one receptacle to the other via the chamber, and   c) the chamber has transversely disposed within it an affinity support having high affinity for a high abundance molecule and an affinity support coupled to one or more avian antibodies directed against the high abundance molecule.   
     
     
         26 . A device according to  claims 25 , in which the antibody is a chicken antibody. 
     
     
         27 . A device according to  claim 26 , in which the antibody is chicken IgY. 
     
     
         28 . A device according to  claim 25 , in which the antibody is a first generation avian polyclonal antibody. 
     
     
         29 . A device according to any  claim 25 , in which the antibody is a second generation avian polyclonal antibody. 
     
     
         30 . A device according to  claim 25 , in which the high abundance molecule is one present in serum or plasma. 
     
     
         31 . A device according to  claim 30 , in which the high abundance molecule is albumin or immunoglobulin. 
     
     
         32 . A device according to  claim 25 , in which the receptacles are hypodermic syringes and the chamber is a Luer-type cartridge. 
     
     
         33 . A device according to  claim 25 , in which the chamber is adapted to couple directly to a separation or analytical apparatus. 
     
     
         34 . A kit for depletion of a high-abundance molecule from a biological sample, comprising:
 a) a first affinity support with high affinity for the high-abundance molecule; and   b) a second affinity support coupled to an avian antibody directed against the high-abundance molecule.   
     
     
         35 . A kit according to  claim 34 , in which the antibody is a chicken antibody. 
     
     
         36 . A kit according to  claim 35 , in which the antibody is chicken IgY. 
     
     
         37 . A kit according to  claim 34 , in which the antibody is a first generation avian polyclonal antibody. 
     
     
         38 . A kit according to  claim 34 , in which the antibody is a second generation avian polyclonal antibody. 
     
     
         39 . A kit according to  claim 34 , in which the high abundance molecule is one present in serum or plasma. 
     
     
         40 . A kit according to  claim 39 , in which the high abundance molecule is albumin or immunoglobulin. 
     
     
         41 . A kit according to  claim 34 , comprising a chamber having two openings, in which
 a) each opening is adapted to fit sealingly to a receptacle,   b) the sample can be transferred from one receptacle to the other via the chamber, and   c) the chamber has transversely disposed within it an affinity support having high affinity for a high abundance molecule and an affinity support coupled to one or more avian antibodies directed against the high abundance molecule.   
     
     
         42 . A kit according to  claim 34 , comprising a diluent suitable for use with biological fluids.