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Detection of mouse alloantibodies by rosetting with protein A-coated sheep red blood cells
Staphylococcal protein A has an affinity for the Fc portion of the IgG molecule of different species and can therefore be used to detect cell-bound immunoglobulin. Using this property, protein A coupled to sheep red blood cells via chromic chloride can detect alloantibodies to mouse H-2, Thy-1, Ly-1, 2, 4, 5, 6, and 7, and Ia antigenic specificities bound to the surface of lymphocytes by the formation of rosettes. In comparison with other rosetting and cytotoxicity assays, the protein A assay shows a greater sensitivity than does cytotoxicity using spleen cells as the target, as does the sheep anti-mouse Ig rosetting assay, whereas cytotoxicity shows greater sensitivity with some antisera on thymocytes. The major advantages of the protein A assay are that constant low reproducible backgrounds are obtained, there is no need to remove surface Ig by capping prior to antiserum treatment, and that viable cells can be recovered.
Staphylococcal protein A has an affinity for the Fc portion of the IgG molecule of different species and can therefore be used to detect cell-bound immunoglobulin. Using this property, protein A coupled to sheep red blood cells via chromic chloride can detect alloantibodies to mouse H-2, Thy-1, Ly-1, 2, 4, 5, 6, and 7, and Ia antigenic specificities bound to the surface of lymphocytes by the formation of rosettes. In comparison with other rosetting and cytotoxicity assays, the protein A assay shows a greater sensitivity than does cytotoxicity using spleen cells as the target, as does the sheep anti-mouse Ig rosetting assay, whereas cytotoxicity shows greater sensitivity with some antisera on thymocytes. The major advantages of the protein A assay are that constant low reproducible backgrounds are obtained, there is no need to remove surface Ig by capping prior to antiserum treatment, and that viable cells can be recovered.