Secondary Antibody Handbook
IHC Primary Antibody Selection & Optimization
This technology brief demonstrates the advantage of using horseradish peroxidase (HRP) polymer conjugated secondary antibodies for sensitive and accurate detection of antigens by immunohistochemistry (IHC). HRP polymer conjugates address the major limitations of biotin-based IHC detection systems, reducing background staining and eliminating extra steps.
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Traditional immunohistochemistry (IHC) methods are often inadequate when detecting low abundance antigens or under conditions of non-optimal antibody-antigen binding. Using low affinity primary antibodies or targeting epitopes that are difficult to detect may lead to poor or inconsistent antigen staining. Several signal amplification methods have been developed to improve assay sensitivity, but these methods commonly result in high background staining. Here we show an advanced method that uses a horseradish peroxidase (HRP)-polymer conjugate to dramatically increase the lower limit of detection without significantly contributing to background signal. Compared to the avidin-biotin complex (ABC) detection system, the polymer-based amplification method reduces the amount of primary antibody needed by 3-fold and shortens the incubation period from
overnight to one hour. These results demonstrate the superior performance of the polymer-based signal amplification method over the ABC method in immunohistochemical analysis of target protein expression.