Isolation of a soluble 42 KD pancreatic islet cell autoantigen
AssigneeExocell, Inc.
InventorsMargo P. Cohen, Van-Yu Wu
Life of this patent
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Abstract
A method of isolating soluble pancreatic islet antigen employs extraction with a non-ionic detergent and fractionation on a gel filtration column. Fractions are screened for high immunoreactivity with diabetic serum which is positive for ICA immunofluorescence when tested on tissue sections of a pancreas from a human blood group O donor. The isolated soluble antigen is suitable for measuring autoantibodies in both complement fixation assays as well as ELISA-type and immunoblotting formats. Methods of using isolated soluble antigen to test serum for autoantibodies are also taught.
Claims27
A cell-free antigenic preparation useful for measuring autoantibody titers in blood, said preparation comprising a detergent-soluble antigen which immunologically binds to circulating autoantibodies to pancreatic islet cells associated with diabetes mellitus, wherein said preparation immunologically binds at least two-fold less to antibodies in serum from non-diabetic humans than to antibodies in serum from diabetic humans, wherein said antigen has a molecular weight of between 40 and 45 kD when measured on SDS-polyacrylamide gels, wherein said antigen is obtainable from pancreases of humans having an HLA type selected from the group consisting of HLA-DR 7;
- HLA-DR 1, w-11;
- HLA-DR 11,7;
- and HLA-DR 2,4, and wherein said antigen is substantially purified from other antigens which are not immunologically reactive with diabetes-specific, islet cell antibodies.
A cell-free antigenic preparation useful for measuring autoantibody titers in blood, said preparation comprising a detergent-soluble antigen which immunologically binds to circulating autoantibodies to pancreatic islet cells associated with diabetes mellitus, wherein said preparation immunologically binds at least two-fold less to antibodies in serum from non-diabetic humans than to antibodies in serum from diabetic humans, said antigen being substantially purified from other antigens which are not immunologically reactive with diabetes-specific, islet cell antibodies, said antigen having a molecular weight of between 40 and 45 Kd when measured on SDS-polyacrylamide gels, said antigen being obtainable from pancreases of humans having an HLA type selected from the group consisting of HLA-DR 7;
- HLA-DR 1, w-11;
- HLA-DR 11,7;
- and HLA-DR 2,4, said antigenic preparation made by the process of:
- lysing and extracting islet cells from human pancreas to provide a cell-free extract containing soluble islet cell autoantigen;
- fractionating components of the cell-free extract to provide fractionated soluble components;
- testing the fractionated soluble components in denatured state for immunological binding to circulating autoantibodies to pancreatic islet cells associated with diabetes mellitus;
- selecting the fractionated soluble components with maximum immunological binding in the denatured state.
A method for isolating a detergent soluble autoantigen which is capable of immunologically binding to circulating autoantibodies to pancreatic islet cells associated with diabetes mellitus, wherein said autoantigen immunologically binds at least two-fold less to antibodies in serum from non-diabetic humans than to antibodies in serum from diabetic humans, said autoantigen being substantially purified from other antigens which are not immunologically reactive with diabetes-specific islet cell antibodies, said autoantigen having a molecular weight of between 40 and 45 kD when measured on SDS-polyacrylamide gels, said antigen being obtainable from pancreases of humans having an HLA type selected from the group consisting of HLA-DR 7;
- HLA-DR 1, w-11;
- HLA-DR 11,7;
- and HLA-DR 2,4, said method comprising:
- lysing and extracting islet cells from human pancreas to provide a cell-free extract containing said soluble islet cell autoantigen;
- fractionating components of the cell-free extract to provide fractionated soluble components;
- testing the fractionated soluble components in the denatured state for immunological binding to circulating autoantibodies to pancreatic islet cells associated with diabetes mellitus;
- selecting the fractionated soluble components with maximum immunological binding in the denatured state.
Dependencies are inferred from the claim text.