The Peroxidase-Antiperoxidase (PAP) Method

  • Gary L. Bratthauer
Protocol
Part of the Methods in Molecular Bilogy book series (MIMB, volume 34)

Abstract

The peroxidase-antiperoxidase (PAP) method was pioneered by Sternberger in 1979 (1). The method uses an immunological sandwich amplification and the enzyme peroxidase to effect a signal. The unique feature of this procedure is the enzyme-antibody solution, the PAP immune complex. The horseradish peroxidase enzyme, itself an immunogenic protein, is used to inoculate a given species, and a polyclonal immune response is generated against the enzyme. This antiserum is harvested and placed in solution with the enzyme so that immune complexes form that remain soluble. These complexes form with a molar ratio of two molecules of IgG to three molecules of enzyme. Furthermore, not only does this complex remain soluble, but the enzymatic activity of the peroxidase is not affected by the attached immunoglobulins. The antiperoxidase antibodies are from the same species as that which has produced a primary antibody raised against a tissue antigen. These two antibodies, one directed against the tissue antigen and the other directed against the peroxidase enzyme, can be linked by another antibody raised in an alternate species against immunoglobulin from the first species (seeFig. 1).

References

  1. 1.
    Sternberger, L. (1979) Immunocytochemistry, 2nd ed. Wiley, New York.Google Scholar
  2. 2.
    Cerio, R. and MacDonald, D (1988) Routine diagnostic immunohistochemical labeling of extracellular antigens in formol saline solution—fixed, paraffinembedded cutaneous tissue J. Am. Acad. Dermatol. 19, 747–753.PubMedCrossRefGoogle Scholar
  3. 3.
    Clements, J and Beitz, A. (1985) The effects of different pretreatment conditions and fixation regimes on serotonin immunoreactivity: a quantitative light microscopic study. J. Histochem. Cytochem. 33, 778–784PubMedGoogle Scholar
  4. 4.
    Laitinen, L., Laitinen, A, Panula, P., Partanen, M., Tervo, K., and Tervo, T. (1983) Immunohistochemical demonstration of substance P in the lower respiratory tract of the rabbit and not of man Thorax 38, 531–536.PubMedCrossRefGoogle Scholar
  5. 5.
    Hsu, S. and Soban, E (1982) Color modification of diaminobenzidine (DAB) precipitation by metallic ions and its application for double immunohistochemistry. J. Histochem. Cytochem 30, 1079PubMedGoogle Scholar
  6. 6.
    Ordronneau, P., Lindstrom, P., and Petrusz. P. (1981) Four unlabeled antibody bridge techniques: A comparison. J. Histochem. Cytochem. 29, 1397–1404.PubMedGoogle Scholar

Copyright information

© Humana Press Inc. 1994

Authors and Affiliations

  • Gary L. Bratthauer
    • 1
  1. 1.Immunopathology DivisionArmed Forces Institute of PathologyWashington

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