Abstract
The peroxidase-antiperoxidase (PAP), avidinbiotin-peroxidase complex (ABC) and catalyzed signal amplification (CSA) methods have been used for quantitative or semiquantitative immunohistochemical analysis. It is however unclear whether intensity of immunostaining in sections amplified by using the PAP, ABC or CSA method reflects an antigen content. Thus, relationship between the intensity and the content was examined in antigen-immobilized nitrocellulose filters or serial sections stained by using the PAP, ABC or CSA method. In the present study, we selected cytochromes P-450 2B1/2 (CYP2B1/2), because the immunostaining intensity of the enzymes detected with the indirect immunoperoxidase (IPO) method under saturation conditions is shown to be proportional to the antigen content in sections. For the IPO method, the intensity in filters or sections increased with increasing concentration of anti-CYP2B1/2 antibody, and then saturated. However, a bell-shaped relationship was observed between the intensity and the primary antibody concentration in filters or sections stained by using the PAP, ABC or CSA method. Furthermore, the amplified intensity in filters or sections was disproportionate 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 suitable for the measurement of antigen content in sections by quantitative immunohistochemistry.
