Amplification of immunostaining intensity by the peroxidase-antiperoxidase, avidin-biotin-peroxidase complex or catalyzed signal amplication method gives erroneous antigen content in sections

The peroxidase-antiperoxidase (PAP), avidin-biotin-peroxidase complex (ABC) and catalyzed signal amplification (CSA) methods have been used for quanti tative or semiquantitative immunohistochemical analysis. It is however uncl ear whether intensity of immunostaining in sections amplified by using the PAP, ABC or CSA method reflects an antigen content. Thus, relationship betw een the intensity and the content was examined in antigen-immobilized nitro cellulose filters or serial sections stained by using the PAP, ABC or CSA m ethod. In the present study, we selected cytochromes P-450 2B1/2 (CYP2B1/2) , because the immunostaining intensity of the enzymes detected with the ind irect immunoperoxidase (IPO) method under saturation conditions is shown to be proportional to the antigen content in sections. For the IPO method, th e intensity in filters or sections increased with increasing concentration of anti-CYP2B1/2 antibody, and then saturated. However, a bell-shaped relat ionship was observed between the intensity and the primary antibody concent ration in filters or sections stained by using the PAP, ABC or CSA method. Furthermore, the amplified intensity in filters or sections was disproporti onate to the antigen content. Thus, erroneous CYP2B1/2 content was obtained from sections in which the intensity was amplified by the PAP, ABC or CSA method. It is therefore unlikely that the PAP, ABC and CSA methods are suit able for the measurement of antigen content in sections by quantitative imm unohistochemistry.