A comparative study of the peroxidase-antiperoxidase method and an avidin-biotin complex method for studying polypeptide hormones with radioimmunoassay antibodies

Am J Clin Pathol. 1981 May;75(5):734-8. doi: 10.1093/ajcp/75.5.734.


A highly sensitive immunoenzymatic technic is presented. The method involves three sequential steps: (1) primary antibody, (2) biotin-labeled secondary antibody, and (3) avidin-biotin-peroxidase complex. Avidin, an egg white protein, has four binding sites for the low-molecular-weight vitamin biotin. Many moieties of biotin can be coupled to the peroxidase molecule. Thus, since a relatively large amount of avidin is incubated with biotin-labeled peroxidase, avidin serves as a link between biotin-peroxidase molecules; in turn, biotin-peroxidase serves as a link between avidin molecules. Consequently, this large lattice-like complex with biotin-binding capability can be attracted to the sites of biotin-labeled antibody, producing a superior staining sensitivity. Several commercially available radioimmunoassay antibodies (e.g., antiglucagon, prolactin, gastrin, growth hormone, and thyroid-stimulating hormone antibodies) were tested for immunohistochemical staining. The unlabeled antibody peroxidase-antiperoxidase method fails to stain gastrin or thyroid-stimulating secretory cells when using these antibodies, and a relatively high antibody concentration is required to produce a positive reaction for glucagon, prolactin, and growth hormone. In contrast, the avidin-biotin-peroxidase complex method successfully demonstrates polypeptide hormones even when antibodies are diluted 20 to 40 times.

Publication types

  • Comparative Study

MeSH terms

  • Antibodies / analysis*
  • Avidin*
  • Biotin*
  • Gastrins / immunology
  • Glucagon / immunology
  • Growth Hormone / immunology
  • Hormones / immunology*
  • Humans
  • Immunoenzyme Techniques / methods*
  • Methods
  • Ovalbumin* / analogs & derivatives
  • Prolactin / immunology
  • Radioimmunoassay*
  • Staining and Labeling
  • Thyrotropin / immunology


  • Antibodies
  • Gastrins
  • Hormones
  • Avidin
  • Biotin
  • Prolactin
  • Thyrotropin
  • Growth Hormone
  • Ovalbumin
  • Glucagon