Multilayer peroxidase-labeled antibody method: comparison with labeled streptavidin-biotin method, avidin-biotin-peroxidase complex method, and peroxidase-antiperoxidase method.
A new, extremely sensitive, "multilayer peroxidase-labeled antibody method" is introduced. This method utilizes peroxidase-labeled antibodies that immuno-react with their antigen, but also can be an antigen to other peroxidase-labeled antibodies. An example of this method is: use rabbit IgG as the first antibody, peroxidase-labeled goat antirabbit IgG as the second antibody, peroxidase-labeled rabbit antigoat IgG as the third antibody, peroxidase-labeled goat antirabbit IgG as the fourth antibody, and peroxidase-labeled rabbit antigoat IgG as the fifth antibody in succession. We compared the sensitivities of this multilayer peroxidase-labeled antibody method with that of commercially available immuno-peroxidase staining kits, i.e., avidin-biotin-complex method and peroxidase-antiperoxidase method, both by immunostaining and enzyme-linked immunosorbent assay. The generated signal and background in immunostained sections were determined by a computer-assisted image analyzer and by enzyme-linked immunosorbent assay spectrophotometrically. It was found that a maximum signal/background ratio was obtained with those sections reacted with the first four antibodies in succession by the multilayer peroxidase-labeled antibody method, and those reacted with the five antibodies in succession by the multilayer peroxidase-labeled antibody method in the enzyme-linked immunosorbent assay. Thus, the multilayer peroxidase-labeled antibody method is more sensitive than the avidin-biotin-complex method and the peroxidase-antiperoxidase method.
Shin
M
M
Department of Anatomy III, Nagasaki University School of Medicine, Japan.
Izumi
S
S
Nakane
P K
PK
D000818:Animals; D001360:Avidin; D001426:Bacterial Proteins; D001710:Biotin; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods / Q000706:statistics & numerical data; D005069:Evaluation Studies as Topic; D007124:Immunoenzyme Techniques / Q000706:statistics & numerical data; D007150:Immunohistochemistry / Q000379:methods / Q000706:statistics & numerical data; D008099:Liver / Q000378:metabolism; D008297:Male; D010544:Peroxidases; D011817:Rabbits; D051381:Rats; D017208:Rats, Wistar; D012680:Sensitivity and Specificity; D019809:Streptavidin
D001426:Bacterial Proteins; D001360:Avidin; D001710:Biotin; D019809:Streptavidin; D010544:Peroxidases