Final answer:
You cannot bind fluorophores directly to the primary antigen in IFAs because it may not provide a strong enough signal. Using secondary antibodies increases the number of attached fluorophores and amplifies the signal, facilitating better visualization of the target antigen.
Step-by-step explanation:
The reason why you can't just bind the fluorophores to the primary antigen directly in immunofluorescence assays (IFA) is due to the desire for amplification of the signal. If fluorophores are bound directly to the primary antibodies, which are then attached to the antigen, the signal may not be strong enough for detection.
This is especially relevant when the amounts of antigen are very low. On the other hand, using a secondary antibody that binds to the primary antibody allows for multiple fluorescently-labelled secondary antibodies to attach to each primary antibody, thereby significantly increasing the fluorescent signal, or amplifying it.
In direct fluorescent antibody tests (DFA), the fluorescent antibody binds directly to the antigens of interest that may be bacterial or viral pathogens in clinical samples. This approach is simpler but less sensitive than IFA.
In contrast, indirect immunofluorescence uses secondary antibodies that bind to the primary antibodies that have attached to antigens, such as Treponema cells in clinical settings.
This method enhances detection as each primary antibody can be bound by multiple fluorescent secondary antibodies, thus making the pathogen cells easier to visualize.