Self-sandwich Method. An Improved Immunoperoxidase Technic for the Detection of Small Amounts of Antigens

Abstract

The unlabeled antibody peroxidase-antiperoxidase (PAP) sequence has been widely accepted as the most sensitive method for demonstrating antigens in paraffin sections. However, the results have been unpredictable in routine surgical specimens, with frequent false-negative stains. In order to amplify the staining reactions a self-sandwich method was used. The amplifier (antigen itself) was added after the application of specific antibody. The procedure may be repeated several times before the application of the bridge antiserum followed by PAP complexes. The method was designed to increase the number of antigen-antibody layers without increasing the number of heteroantisera in the system. Specimens derived from routine tonsillectomy were studied semiquantitatively. Based on the number of positively stained Igcontaining cells in germinal centers, the sensitivity of the selfsandwich method was estimated to be 20 to 50 times that of the PAP method. In addition, extracellular Ig (reticular staining) and surface Ig were also stained positively in the germinal centers and lymphocytic mantles, respectively, but were not demonstrated by the PAP method. The enhancement of the sensitivity was achieved without compromising the specificity.