A comparative study of the peroxidase-antiperoxidase method and an avidin-biotin complex method for studying polypeptide hormones with radioimmunoassay antibodies.
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.
D000906:Antibodies / Q000032:analysis*; D001360:Avidin; D001710:Biotin; D005755:Gastrins / Q000276:immunology; D005934:Glucagon / Q000276:immunology; D013006:Growth Hormone / Q000276:immunology; D006728:Hormones / Q000276:immunology*; D006801:Humans; D007124:Immunoenzyme Techniques / Q000379:methods*; D008722:Methods; D010047:Ovalbumin / Q000031:analogs & derivatives; D011388:Prolactin / Q000276:immunology; D011863:Radioimmunoassay; D013194:Staining and Labeling; D013972:Thyrotropin / Q000276:immunology
D000906:Antibodies; D005755:Gastrins; D006728:Hormones; D001360:Avidin; D001710:Biotin; D011388:Prolactin; D013972:Thyrotropin; D013006:Growth Hormone; D010047:Ovalbumin; D005934:Glucagon