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 ΔTth 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 (PrPsc) 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. PrPsc detection was consistent using all three methods. The clearest signals were obtained using the indirect immunoAT method, suggesting significant potential for this method.