A STRATEGY FOR IMMUNOHISTOCHEMICAL SIGNAL ENHANCEMENT BY END-PRODUCT AMPLIFICATION

We report a novel strategy, called end-product (EP) amplification, cap able of enhancing the sensitivity of immunohistochemical procedures by about an order of magnitude or more. The strategy employs an antibody (anti-EP) to the product generated by the action of horseradish perox idase on 3,3'-diaminobenzidine (DAB), and can be extended to the produ cts of other enzymes as well, e.g., alkaline phosphatase, Amplificatio n is the consequence of the ability of anti-EP to detect the multiplic ity of product moelcules resulting from the turnover of substrate by a single enzyme molecule, The subsequent detection of anti-EP was by bi otinylated goat anti-rabbit antibody, followed by avidin-peroxidase an d DAB or by avidin-alkaline phosphatase and Vector Red, Further amplif ication can be accomplished by repeated cycles of the protocol, Anti-E P was produced by immunization with a bovine serum albumin (BSA) conju gate of a soluble polymer of DAB, prepared by a carefully controlled r eaction of DAB with horseradish peroxidase and hydrogen peroxide, Coup ling to BSA (and to RSA) was accomplished with glutaraldehyde, The tit er of anti-EP was established by ELISA. Formalin-fixed, paraffin-embed ded sections of five cases of Hodgkin's disease and five tonsils with follicular hyperplasia were immunolabeled for the following lymphoid m arkers: CD3, CD20, CD30, CD45RA, and CD68, EP amplification with anti- EP was also applied to cases of CMV pneumonia and cerebral toxoplasmos is to determine whether this procedure could improve detection of the infectious agents, Immunolabeling of the primary antibody was performe d by the avidin-biotin-peroxidase technique with DAB as the reaction s ubstrate, The specificity of EP amplification was tested by demonstrat ing binding of anti-EP with Vector Red with the generation of a fluore scence end-point, There was complete congruence in the distribution of the DAB signal and the red immunofluorescence representing EP amplifi cation. The intensity of the DAB signal was increased as much as Iii-f old by EP amplification, making possible a reduction in the amount of the primary antibody by as much as 85-90%, Sensitivity also increased with respect to weakly expressed antigens and low concentrations of in fectious agents.