Preparation Method

Abstract

A method of performing an immunologic evaluation of a subject, comprising collecting a whole blood sample from the subject, maintaining the sample for at least 6 hours after the collection, purifying a population of cells comprising lymphocytes and antigen presenting cells from the maintained sample, optionally by a process incorporating a positive or negative affinity selection step, and using the purified cells in a cell-mediated immunoassay (CMI assay).

1 . A method of performing a cell-mediated immunoassay, comprising:
 (i) collecting a whole blood sample from said subject;   (ii) maintaining said sample for at least 6 hours after said collection,   (iii) purifying cells comprising lymphocytes and antigen presenting cells from said maintained sample by a process comprising a positive or negative affinity selection step; and   (iv) using said purified cells in a cell-mediated immunoassay.   
   
   
       2 . The method of  claim 1 , wherein said sample is maintained for at least 10 hours. 
   
   
       3 . The method of  claim 2 , wherein said sample is maintained for at least 12 hours. 
   
   
       4 . The method of  claim 3 , wherein said sample is maintained between 12 and 36 hours, 12 to 48 hours, or 24 to 48 hours. 
   
   
       5 . The method of  claim 1 , wherein said sample is maintained between 2-8° C. 
   
   
       6 . The method of  claim 1 , wherein said sample is maintained between 18-25° C. 
   
   
       7 . The method of  claim 1 , wherein said process comprises a negative affinity selection step to remove granulocytes from said maintained sample. 
   
   
       8 . The method of  claim 7 , wherein said process further comprises contacting said maintained sample with an antibody preparation comprising anti-CD66b and glycophorin A antibodies to aggregate red blood cells and granulocytes in said maintained sample, and removing said aggregated red blood cells and granulocytes from said maintained sample by centrifugation. 
   
   
       9 . The method of  claim 7 , wherein said granulocytes are removed by contacting said maintained sample with a solid support comprising an anti-CD15 ligand to remove CD15+ cells from said maintained sample. 
   
   
       10 . The method of  claim 9 , wherein said solid support further comprises magnetic beads, and wherein said process further comprises contacting said maintained sample with said beads to bind CD15+ cells to said beads, and separating said beads having said CD15+ cells bound thereto from said maintained sample. 
   
   
       11 . The method of  claim 9 , wherein red blood cells in said maintained sample are removed by lysis or by filtration. 
   
   
       12 . The method of  claim 1 , wherein said purification leads to an increase in said maintained sample of both the ratio of T cells to total cells and the ratio of antigen presenting cells to total cells. 
   
   
       13 . The method of  claim 1 , wherein said sample is not frozen before said purification. 
   
   
       14 . The method of  claim 1 , wherein said purified cells are not frozen before said use. 
   
   
       15 . The method of  claim 1 , wherein said process comprises a positive affinity selection step. 
   
   
       16 . The method of  claim 15 , wherein said positive affinity selection step selects all types of T cells. 
   
   
       17 . The method of  claim 15 , wherein said process further comprises contacting said maintained sample with a solid support having attached thereto ligands, wherein said ligands bind to cell surface proteins present on the surface of said lymphocytes and antigen presenting cells, and wherein said lymphocytes and antigen presenting cells are bound to said solid support via said ligands. 
   
   
       18 . The method of  claim 17 , wherein said solid support comprises one or more ligands selected from anti-CD4, anti-CD8, anti-CD19 and anti-CD14 ligands to purify CD4+, CD8+, CD19+ or CD 14+ cells from said maintained sample. 
   
   
       19 . The method of  claim 17 , wherein said bound lymphocytes and antigen presenting cells are separated from said solid support before said use. 
   
   
       20 . The method of  claim 17 , wherein said bound lymphocytes and antigen presenting cells are retained on said solid support for said use. 
   
   
       21 . The method of  claim 17 , wherein said solid support comprises magnetic beads. 
   
   
       22 . The method of  claim 21 , wherein each said magnetic bead has bound thereto anti-CD4, anti-CD8 and anti-CD19 ligands. 
   
   
       23 . The method of  claim 21 , wherein each magnetic bead has bound thereto one or more ligands selected from anti-CD4, anti-CD8 and anti-CD19 ligands, and wherein said beads are mixed together to provide said solid support for binding to CD4+, CD8+ and CD 19+ cells. 
   
   
       24 . A method of performing a cell-mediated immunoassay, comprising
 (i) collecting a whole blood sample from said subject,   (ii) diluting said whole blood sample with cell growth medium,   (iii) maintaining said diluted sample for at least 6 hours after said collection,   (iv) purifying cells comprising lymphocytes and antigen presenting cells from said maintained sample;   (iv) using said purified cells in a cell-mediated immunoassay (CMI assay).   
   
   
       25 . The method of  claim 24 , wherein said whole blood sample is diluted with said cell growth medium in a ratio of 1:1. 
   
   
       26 . The method of  claim 24 , wherein said cell growth medium is AIM-V. 
   
   
       27 . The method of  claim 1 , said CMI assay is an ELISPOT assay. 
   
   
       28 . The method of  claim 27 , wherein said lymphocytes are T cells, and wherein said purified T cells maintain antigen specific cytokine response in said ELISPOT assay. 
   
   
       29 . The method of  claim 28 , wherein said process comprises a negative affinity selection step to remove granulocytes from said maintained sample. 
   
   
       30 . The method of  claim 1 , further comprising removing red blood cells from said maintained sample by filtration, wherein said CMI assay is an ELISPOT assay. 
   
   
       31 . The method of  claim 30 , further comprising passing said whole blood sample through a filter under conditions allowing said red blood cells to pass through said filter and allowing said lymphocytes and antigen presenting cells to bind to said filter, and recovering said bound lymphocytes and antigen presenting cells from said filter. 
   
   
       32 . The method of  claim 31 , wherein said filter is back flushed to recover said lymphocytes and antigen presenting cells. 
   
   
       33 . The method of  claim 1 , wherein said subject is a human. 
   
   
       34 . The method of  claim 33 , wherein said CMI assay is to diagnose or monitor a disease. 
   
   
       35 . A kit for carrying out the method of  claim 1 , comprising:
 (a) a cytokine specific antibody, and   (b) one or more of ligands selected from anti-CD15, anti-CD4, anti-CD8, anti-CD19 and anti-CD14 ligands.   
   
   
       36 . The kit of  claim 35 , further comprising instructions for carrying out said method. 
   
   
       37 . The kit of  claim 35 , further comprising instructions for carrying out an ELISPOT assay. 
   
   
       38 . The kit of  claim 35 , wherein said cytokine specific antibody is attached to a microtiter plate. 
   
   
       39 . The kit of  claim 35 , wherein said one or more ligands are bound to a solid support.