Allo and Auto-Reactive T-Cell Epitopes

Abstract

The present invention relates to a pharmaceutical composition for the prevention of alloimmunisation of a subject or the immunosuppression of a response elicited by alloimmunisation of a subject or an autoimmune haemolytic disease for said composition comprising an immunologically effective epitope of a rhesus protein or an immunologically active analogue or derivative thereof.

1 - 23 . (canceled) 
     
     
         24 : A method for determining effect of one or more epitopes from a rhesus protein on a human lymphocyte, in vitro, comprising:
 (a) stimulating the lymphocyte with one or more epitope/peptide of a rhesus protein;   (b) between 4 to 7 days later resuspending the cultures and transferring aliquots into plates prepared in the following manner:
 (i) coating each well in the plate with monoclonal anticytokine capture antibody; 
 (ii) washing the plate at least once with Hanks Buffered Salt Solution (HBSS); 
 (iii) blocking any non-specific binding using an appropriate solution; 
   (c) incubating the plates with lymphocyte culture for 12-36 hours at 30-40° C. in an atmosphere of substantially 5% CO 2  and substantially 95% air;   (d) washing the plates at least once with Tween/PBS to remove unbound lymphocytes;   (e) introducing an appropriate biotinylated monoclonal detection antibody to each well and incubating for 30-60 minutes at room temperature;   (f) washing the plates at least once with Tween/PBS to remove unbound detection antibody;   (g) introducing ExtrAvidin-alkaline phosphatase conjugate and incubating for 15-45 minutes;   (h) washing the plates at least once with Tween/PBS to remove unbound ExtrAvidin-alkaline phosphatase conjugate;   (i) developing the plates with p-nitrophenyl phosphate in 0.05M carbonate alkaline buffer pH 9.6 added to each well; and   (j) reading the absorbance at 405 nm.   
     
     
         25 : The method according to  claim 24  wherein the rhesus protein is selected from the group consisting of RhD, RhC, Rhc, RhE, Rhe and Rh50 protein. 
     
     
         26 : The method according to  claim 24  wherein the epitope/peptide of a rhesus protein is selected from at lease one of SEQ ID Numbers 2, 5, 6, 11, 12, 14, 28, 29, 31, 38, 39, 44, 47, 50, 51, 66, 75, 77, 78, 79, 81 and 84. 
     
     
         27 : The method according to  claim 24  wherein the epitope/peptide is artificially synthesized. 
     
     
         28 : The method according to  claim 24  wherein the monoclonal anti-cytokine capture antibody is specific for IFN-γ, IL-10 or TGF-β.