Studies on specific anti-macrophage serum (AMS) I) Preparation of specific rabbit anti-guinea pig peritoneal macrophage serum and its antigen analysis, --including distribution of macrophages in the sites of skin delayed hypersensitivity

Macrophage localization in the site of immune response has been investigated using antimacrophage antibody. Specificity of the anti-macrophage sera reported so far has not always been critically evaluated. In the present work, rabbit anti-guinea pig peritoneal macrophage serum (AMS) which reacts specifically with guinea pig macrophages has been successfully prepared by extensive absorptions with various tissues, such as red blood cells, spleen nonadherent cells and kidney homogenate. Specificity of AMS was examined by an immunofluorescent technique and by immunodiffusion. Macrophage specific antigens were analyzed by the sucrose gradient technique. For these, macrophages were fractionated into crude nuclear fraction (M1), crude mitochondrial fraction (M3), crude microsomal fraction (M5) and their supernatants (M4). Each of these fractions was tested by immunodiffusion against AMS with and without absorptions. AMS without absorptions revealed numerous precipitine lines against M1, M3, M4, M5. In contrast, AMS with absorptions showed only one fused precipitin line against M3 and M5. These data, therefore, strongly suggested that the absobed AMS retained antibodies directed at macrophage specific antigens localized in part in the crude mitochondrial and microsomal fractions. Using this specifically prepared AMS, the distributions of macrophages in the site of skin delayed hypersensitivity has also been investigated.