Question

Indirect detection systems and direct detection systems. What do we mean? (Bioinspired Vision Question)

Answer 1

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.