Preparation of Specific Rabbit Anti-Guinea Pig Peritoneal Macrophage serum (AMS) and its Antigen Analysis

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  • Miyake Susumu
    The Third Department of Internal Medicine, Medical School, Okayama University
  • Yamana Seizo
    The Third Department of Internal Medicine, Medical School, Okayama University
  • Nishiya Koji
    The Third Department of Internal Medicine, Medical School, Okayama University
  • Ofuji Tadashi
    The Third Department of Internal Medicine, Medical School, Okayama University

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  • 特異的抗マクロファージ血清の作製
  • トクイテキ コウ マクロファージ ケッセイ ノ サクセイ

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Abstract

Macrophage localization in the site of immune response has been investigated using FITC-labelled anti-macrophage antibody. The specificity of anti-macrophage sera which have been applied so far, has not always been critically evaluated, resulting from the cross reactivity with other cell components. In the present work, rabbit anti-guinea pig serum (AMS) which specifically reacts with guinea pig peritoneal macrophage has been successfully prepared by the absorption with various organs, such as red blood cells, spleen non-adherent cells and kidney homogenate. Specificity was examined by immunofluorescent technique and immunodiffusion. In addition, macrophage specific antigen was further investigated by the sucrose gradient technique, by which macrophages were fractionated into crude nuclear fraction (M1), crude mitochondrial fraction (M3), crude microsomal fraction (M5) and their supernatant (M4). Each of these fractions was tested by immunodiffusion against AMS before and after absorption. AMS before absorption revealed numerous precipitine lines against M1, M3, M5 and M4. In contrast, absorbed AMS demonstrated only one precipitine line against M3 and M5. Precipitine line seen in M3 and M5 was fused. These data, therefore strongly suggested that the absorbed AMS gained high specificity against macrophage specific antigen which localized in the crude mitochondrial and microsomal fractions.

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