Direct vs indirect detection in IHC
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The method of detecting a primary antibody that is bound to the antigen of interest in an IHC experiment can be either direct or indirect.
In direct detection methods, the primary antibody is directly conjugated to a label. During indirect detection, the primary antibody is bound by a labeled secondary antibody that has been raised against the host species of the primary antibody. Indirect methods may also include amplification steps to increase signal intensity. The choice of direct or indirect detection is often prescribed by the expression level of the target antigen.
Direct detection is suitable for detecting highly expressed antigens. For direct detection, the primary antibody can be conjugated to an enzyme, such as horse radish peroxidase (HRP) or alkaline phosphatase (AP), or a fluorochrome. The benefit of direct detection is that an additional incubation step with a secondary reagent is not necessary. Another significant benefit of direct detection is increased flexibility in the design of multicolor experiments, given the wide range of fluorochromes that are available.
Abcam offers a wide range of primary antibodies conjugated to many different labels. Find out more. Our rapid one-step antibody conjugation kits allow you to create your own directly-labeled primary antibodies with 100% antibody recovery. Choose from enzymatic, fluorescent or gold labels. Find out more.
Indirect detection is more suitable for studies of poorly expressed antigens, which benefit from the signal amplification provided by the secondary reagent. Signal amplification occurs through the potential for two or more labeled secondary antibodies to bind to each primary antibody. However, the use of a secondary antibody requires additional blocking steps and controls. The signal may be amplified further by using avidin or streptavidin with biotinylated secondary antibodies. Amplification arises through the ability of each avidin or streptavidin molecule to bind to 4 biotinylated secondary antibodies. However, the use of biotin-based methods requires additional blocking steps to prevent non-specific binding to endogenous biotin. Find out more about biotin-based methods and the next-generation polymer methods here.
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