Method and compositions for specifically detecting physiologically acceptable polymer molecules

Abstract

The present invention relates to a method for determining the amount of a physiologically acceptable polymer molecule bound to a protein, an antibody or other composition being capable of specifically binding to a physiologically acceptable polymer molecule, and a kit containing said antibody or composition.

1 . A method for determining the number of water soluble polymer molecules bound to a protein or protein complex in a polymer-protein conjugate, comprising the steps of
 detecting binding between
 (i) a polymer:protein conjugate having one or more polymers bound to the protein and 
 (ii) an antibody that specifically binds said polymer, said antibody detectable when bound to said polymer:protein conjugate, 
   wherein the number of polymers in the polymer:protein conjugate correlates with levels of antibody detected bound to the polymer:protein conjugate when compared to a known control.   
     
     
         2 . The method of  claim 1 , wherein the antibody comprises a detectable label. 
     
     
         3 . The method of  claim 2 , wherein the detectable label is selected from the group consisting of an enzyme, a radioactive label, a fluorophore, an electron dense reagent, biotin, digoxigenin, haptens, and proteins which are made detectable by addition of any of these labels. 
     
     
         4 . The method of  claim 1 , wherein the polymer:protein conjugate is bound to a carrier matrix prior to binding with the antibody. 
     
     
         5 . The method of  claim 4 , wherein the matrix is selected from the group consisting of a microcarrier, a particle, a membrane, a strip, paper, a film, a bead or a plate. 
     
     
         6 . The method of  claim 4 , wherein the level of antibody detected is measured as absorbance of the detectable label. 
     
     
         7 . The method of  claim 1 , wherein the polymer:protein conjugate is isolated using sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a membrane prior to the detecting. 
     
     
         8 . The method of  claim 7 , wherein the number of polymers in the polymer:protein conjugate is calculated based on the molecular weight of the protein-polymer conjugate compared to a known control. 
     
     
         9 . The method of  claim 7 , wherein the molecular weight of the polymer-protein complex correlates with the protein subunit comprising the polymer molecule. 
     
     
         10 . The method of  claim 1 , wherein the protein or protein complex is a blood clotting factor or a blood clotting factor complex. 
     
     
         11 . The method of  claim 10  wherein the blood clotting factor or blood clotting factor complex is human. 
     
     
         12 . The method of  claim 10  wherein the blood clotting factor is selected from the group consisting of Factor II, Factor III, Factor V, Factor VII, Factor VIII, Factor IX, Factor X, Factor XI, Factor XII, Facter XIII, von Willebrand Factor, protein C and antithrombin III. 
     
     
         13 . The method of  claim 11  wherein the blood clotting factor complex is FactorVIII:VWF. 
     
     
         14 . The method of  claim 1 , wherein the water soluble polymer is releasable. 
     
     
         15 . The method of  claim 1 , wherein the water soluble polymer is hydrolyzable. 
     
     
         16 . The method of  claim 1 , wherein the polymer is selected from the group consisting of poly(alkylene glycol), poly(propylene glycol), copolymers of ethylene glycol and propylene glycol, poly(oxyethylated polyol), poly(olefinic alcohol), poly(vinylpyrrolidone), poly(hydroxyalkylmethacrylamide), poly(hydroxyalkylmethacrylate), poly(saccharides), poly(α-hydroxy acid), poly(vinyl alcohol), polyphosphasphazene, polyoxazoline, and poly(N-acryloylmorpholine). 
     
     
         17 . The method of  claim 1 , wherein the water soluble polymer is polyethylene glycol (PEG) or a derivative thereof. 
     
     
         18 . The method of  claim 17 , wherein the PEG is from 3 to 100 kDa. 
     
     
         19 . The method of  claim 18 , wherein the PEG has a molecular weight in a range of about 5 kDa to about 60 kDa. 
     
     
         20 . The method of  claim 18 , wherein the PEG has a molecular weight in a range of about 5 kDa to about 40 kDa. 
     
     
         21 . The method of  claim 18 , wherein the PEG has a molecular weight in a range of about 5 kDa to about 15 kDa. 
     
     
         22 . The method of  claim 18 , wherein the PEG has a molecular weight in a range of about 5 kDa to about 10 kDa. 
     
     
         23 . A method for determining the number of water soluble polymer molecules bound to a protein or a protein complex comprising,
 contacting said polymer with an antibody that specifically binds said polymer, said antibody detectable when bound to said polymer,
 wherein the number of polymers bound by the antibody correlates with levels of antibody detected bound when compared to a known control. 
   
     
     
         24 . The method of  claim 1  or  23 , wherein the antibody is a polyclonal antibody. 
     
     
         25 . The method of  claim 1  or  23 , wherein the antibody is a monoclonal antibody.