ImmunoAT method: An initial assessment for the detection of abnormal isoforms of prion protein in formalin-fixed and paraffin-embedded tissues.
The AT-tailing method is a labelling technique that utilises oligo(dA-dT)-dependent signal amplification. In this study, a new immunohistochemical application of the immunoAT method was developed. This method uses an oligo(dA-dT)-conjugated primary antibody (direct immunoAT method) or an oligo(dA-dT)-conjugated secondary antibody (indirect immunoAT method). Fifteen-base oligo(dA-dT)-conjugated antibodies (IgG-ATs) were prepared in advance by conjugating maleimide-activated oligo(dA-dT) to IgG via free sulfhydryl residues that had been introduced on the surface of IgG using Traut's reagent. Following the reaction with the target antigen and the IgG-AT, oligo(dA-dT) was elongated by DeltaTth DNA polymerase in the presence of dATP, dTTP and biotinylated dUTP, consequently labelling the antigen-antibody complex with a large amount of biotin. To initially evaluate the immunoAT method, the presence or absence of prion protein (PrP(sc)) was determined in formalin-fixed and paraffin-embedded sections of the medulla oblongata of cattle which had been under active surveillance for bovine spongiform encephalopathy. Sections were examined using direct and indirect immunoAT methods and the EnVision+ system (Dako) under conditions that were identical except for the differing IgG-AT and AT-tailing methods. PrP(sc) detection was consistent using all three methods. The clearest signals were obtained using the indirect immunoAT method, suggesting significant potential for this method.
Sato
Yuko
Y
Department of Pathology, National Institute of Infectious Diseases, Shinjuku-ku, Tokyo 162-8640, Japan. kiyonaga@nih.go.jp
Shimonohara
Nozomi
N
Hanaki
Ken-Ichi
K
Goto
Motoki
M
Yamakawa
Yoshio
Y
Horiuchi
Motohiro
M
Takahashi
Hidehiro
H
Sata
Tetsutaro
T
Nakajima
Noriko
N
D000818:Animals; D000906:Antibodies / Q000378:metabolism; D002417:Cattle; D016643:Encephalopathy, Bovine Spongiform / Q000175:diagnosis*; D005557:Formaldehyde; D007150:Immunohistochemistry / Q000379:methods*; D017403:In Situ Hybridization; D008526:Medulla Oblongata / Q000737:chemistry / Q000473:pathology; D009838:Oligodeoxyribonucleotides / Q000737:chemistry*; D016612:Paraffin Embedding; D011067:Poly dA-dT / Q000737:chemistry*; D018620:PrPSc Proteins / Q000276:immunology / Q000302:isolation & purification*; D016707:Tissue Fixation
D000906:Antibodies; D009838:Oligodeoxyribonucleotides; D018620:PrPSc Proteins; C016023:oligo(dA-dT); D005557:Formaldehyde; D011067:Poly dA-dT