Capture, Concentration And Quantitation Of Abnormal Prion Protein From Biological Fluids Using Depth Filtration

Abstract

Methods for producing biological solutions such as immunoglobulins and in particular anti-D immunoglobulin substantially free of abnormal prion protein resulting therefrom. Specifically provided are methods for aggregation of prions and depth filtration of the biological solution to capture and remove abnormal and if desired, normal prion protein. The prion protein may then be eluted from the depth filter and filter washes and concentrated sufficient for detection at limits currently required by available assays.

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       28 . A method of removing prion protein from whole blood, comprising: 
 (a) clinically centrifuging the blood to separate the red blood cells and platelets therefrom;    (b) decanting supernatant of the centrifugation of step (a) from the red blood cells and platelets;    (c) admixing the supernatant with one or more aggregation aids;    (d) filtering the admixture of step (c) through a membrane or depth filter, thereby removing the prion protein from filtrate; and    (e) adding the red blood cells and platelets separated in step (a) back to the filtrate    
   
   
       29 . The method of  claim 28  wherein the aggregation aids are admixed together or used in series.  
   
   
       30 . The method of  claim 29  wherein the one or more aggregation aids comprise organic solvents of low dielectric constant.  
   
   
       31 . The method of  claim 30  wherein the organic solvents are selected from the group consisting of acetone and water-miscible alcohols.  
   
   
       32 . The method of  claim 30  wherein the organic solvents are selected from the group consisting of ethanol, methanol, isopropyl, isopropanol, n-propanol, isopropyl ether, ketones and aldehydes.  
   
   
       33 . The method of  claim 32  herein the alcohol is ethanol or methanol at a concentration of from about 2% to about 100%.  
   
   
       34 . The method of  claim 29  wherein the one or more aggregation aids are selected from the group containing ammonium sulfate, caprylic acid, trichloroacetic acid (TCA), dialdehydes, heteropoly acids, lactate monohydrate (C18H21N3O 4 H2O), and the metal ions Cu2+, Ni, Zn and Ag.  
   
   
       35 . The method of  claim 28  wherein when the prion protein comprises abnormal prion protein, the one or more aggregation aids are complexing agents.  
   
   
       36 . The method of  claim 35  wherein the complexing agent is selected from the group containing heteropolymolybdates, heteropolytungstates, sodium phosphotungstate (NaPTA), antibodies, enzymes and peptides.  
   
   
       37 . The method of  claim 28  wherein the filter is a membrane or depth filter.  
   
   
       38 . The method of  claim 37  wherein the filter is a depth filter.  
   
   
       39 . The method of  claim 38  wherein the filter has a pore size providing a retention of less than about 6 μm.  
   
   
       40 . The method of  claim 39  wherein the filter has a pore size providing a retention of about 0.6 to about 1.5 microns.  
   
   
       41 . The method of  claim 38  wherein the depth filtration is carried out using a using a depth filter having a pore size providing a retention of less than about 0.6 microns.  
   
   
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       75 . A method of removing prion protein from whole blood, comprising: 
 (a) clinically centrifuging the blood to separate the red blood cells and platelets therefrom;    (b) decanting supernatant of the centrifugation of step (a) from the red blood cells and platelets;    (c) admixing the supernatant with methanol;    (d) filtering the admixture of step (c) through a depth filter having a pore size providing a retention of about 0.6 to about 1.5 microns, thereby removing the prion protein from filtrate; and    (e) adding the red blood cells and platelets separated in step (a) back to the filtrate.