A strategy for immunohistochemical signal enhancement by end-product amplification.

Abstract

We report a novel strategy, called end-product (EP) amplification, capable 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 peroxidase on 3,y-diaminobenzidine (DAB), and can be extended to the products of other enzymes as well, e.g., alkaline phosphatase. Amplification is the consequence of the ability of anti-EP to detect the multiplicity of product moelcula resulting from the tumover of substrate by a single enzyme molecule. The subsequent detection of anti-EP was by biotinylated goat anti-rabbit antibody, followed by avidin-peroxidase and DAB or by avidin-alkaline phosphatase and Vector Red. Further amplification can be accomplished by repeated cydes of the protocol. Anti-EP was produced by immunization with a bovine serum albumin (BSA) conjugate of a soluble polymer of DAB, prepared by a carefully controlled reaction of DAB with horseradish peroxidase and hydrogen peroxide. Coupling to BSA (and to RSA) was accomplished with glutaraldehyde. The titer of anti-EP was established by ELISA.Formalin-fixed, paraffin-embedded sections of five cases of Hodgkin's disease and five tonsils with follicular hyperpla-

Optimizing immunohistochemistry: antigen retrieval and signal amplification

“…The immunogold method with silver enhancement 76 , 77 is also effective. Recently, significant increases in intensity were achieved in a horseradish peroxidase (HRP) technique by using an antibody to the DAB end‐product followed by an ABC detection step 6 .…”

“…An alternative approach to increasing sensitivity is to find better techniques for amplifying the signal, and more sensitive variations of the avidin–biotin complex (ABC) and peroxidase‐antiperoxidase (PAP) techniques have been reported 4 –6 . A powerful recent addition is catalysed reporter deposition (CARD) 7 , 8 , or catalysed signal amplification (CSA), based on peroxidase‐mediated deposition of tyramide bound to biotin or fluorochrome labels, which can then be detected by standard methods.…”

Target and signal amplification: approaches to increase the sensitivity of in situ hybridization

“…1 strategy for signal enhancement by end-product amplification has recently been described by Chen et al (1996). This method, in which anti-DAB antibodies are employed to amplify the signal intensity of conventional DAB-based immunostaining, has been designed for immunohistochemistry but should also be applicable to non-isotopic ISH procedures.…”

Amplification Methods to Increase the Sensitivity of In Situ Hybridization: Play CARD(S)

“…In this way, a lot of extra hapten molecules can be introduced at the hybridization site in situ. Visualization of deposited tyramides can be performed either directly after the CARD End product amplification (anti-DAB antibody strategy) Chen et al 1996 reaction with fluorescence microscopy, if fluorochromelabeled tyramides are used, or indirectly with either fluorescence or brightfield microscopy, if biotin, digoxigenin, or di-or trinitrophenyl are used as haptens, which can act as further binding sites for anti-hapten antibodies or (strept)avidin conjugates (in the case of biotinylated tyramides) (Speel et al 1995b). In addition, fluorescein and rhodamine can be used as haptens, because specific antibodies against these fluorochromes are commercially available from several companies.…”