Visualization of antigen on nitrocellulose membrane by the oxidative coupling reaction of N,N'-dimethyl-p-phenylenediamine and 4-chloro-1-naphthol.
A method which allows the highly sensitive and simple immunodetection of antigen-antibody complexes on nitrocellulose papers has been developed. The method is a modification of the procedure known as the "Nadi reaction" (oxidative coupling of 1-naphthol and N,N'-dimethyl-p-phenylendiamine) for histochemical purposes. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrophoretically transferred to nitrocellulose membranes. Bound proteins were first reacted with a primary antibody and then with a horseradish peroxidase-labeled second antibody. The antigen-antibody complexes on the membranes were visualized with the oxidative coupling solution containing N,N'-dimethyl-p-phenylendiamine and 4-chloro-1-naphthol. Sensitivity was enhanced 4 to 16 times by the new method relative to that of the 4-chloro-1-naphthol method or the 3,3'-diaminobenzidine method.
Kobayashi
R
R
Department of Biochemistry, Akita University School of Medicine, Japan.
Tashima
Y
Y
D015100:3,3'-Diaminobenzidine; D000818:Animals; D000941:Antigens / Q000032:analysis*; D002645:Chickens; D003101:Collodion; D006801:Humans; D007158:Immunologic Techniques; D008567:Membranes, Artificial; D009284:Naphthols; D010084:Oxidation-Reduction; D010655:Phenylenediamines; D011817:Rabbits; D013194:Staining and Labeling / Q000379:methods
D000941:Antigens; D008567:Membranes, Artificial; D009284:Naphthols; D010655:Phenylenediamines; D015100:3,3'-Diaminobenzidine; C025917:4-chloro-1-naphthol; C004781:dimethyl-4-phenylenediamine; D003101:Collodion