In ELISA, your first antibody (primary antibody) binds to the antigen of interest in your sample. The secondary antibody, which is a different type of antibody from the first one, recognizes the first antibody and attaches to it. This second (secondary) antibody connects to a third kind of molecular marker that is visible or can be made visible in some way. This means that all you need are these two kinds of antibodies to achieve the same thing. The primary and secondary antibodies bind, forming a bridge between your antigen and a detection method (such as an enzyme). When you don't know exactly what kind of molecules are present in your sample, indirect ELISA gives you more options:
In ELISA, your first antibody (primary antibody) binds to the antigen of interest in your sample. The primary antibody is conjugated with a fluorescent marker, so you can see it binding to the antigen.
The secondary or detection antibody will bind to any free-floating antigens that were not bound by the primary antibodies. These unbound antigens are then washed away and only those bound by both primary and secondary antibodies remain on your plate for detecting via an enzyme-linked immunosorbent assay (ELISA).
The secondary antibody, which is a different type of antibody from the first one, recognizes the first antibody and attaches to it.
The secondary antibody can be labeled with a dye (such as fluorescein or horseradish peroxidase). The dye in turn can be detected by an enzymatic reaction (using peroxidase) or may be fluorescent and detected by direct viewing under a microscope or by using a special instrument that detects fluorescent proteins such as FITC.
This third kind of molecular marker can be made visible in one of two ways: either by attaching a colored dye or enzyme that changes color when it is added to the sample.
In addition, the secondary antibody can bind to more than one primary antibody, which means that all you need are these two kinds of antibodies to achieve the same thing.
The secondary antibody connects with a third kind of molecular marker that is visible or can be made visible in some way. This can take the form of any number of physical characteristics, such as color changes or fluorescence. The point is that this third type of molecule has something in common with both other types (primary and secondary antibody).
When the primary antibody binds to your antigen, it creates a bridge between the two. This bridge is formed by an enzyme-labelled secondary antibody that has been attached to your antigen. The enzyme-labelled secondary antibody is specific to the primary antibody (i.e., it only binds with one specific type of molecule) and can be detected using a variety of techniques, including colorimetric methods (such as ELISA or immunohistochemistry).
In some cases, you may not know what molecules are present in your sample. In these cases, indirect ELISA is a good choice because it gives you more options. You can use different kinds of antibodies and detection methods to find out what's there. The benefit of using secondary antibodies is that they can identify the specific location of an antigen on a molecule or cell.
In addition to being able to see if proteins are present in your sample and how they're expressed (whether they're free or attached), indirect ELISAs give you additional information about what kind of protein it is and where it's located on the cell surface or within cells themselves.<
Indirect ELISA is a variation of the direct ELISA where the primary antibody is replaced by an enzyme-linked secondary antibody. It may be useful when you have limited amounts of sample available, since there is no need to conjugate a second labeled antigen. Instead, an unbound primary antibody binds to the antigen on your plate, and this bound material can be detected using an enzyme-linked secondary antibody that has been raised against the species for which you want to detect specific antibodies in your samples. The enzyme reacts with its substrate (a chemical that changes color or produces light) in order to produce visible signals from which you can determine whether or not there are specific antibodies present in your samples.
Hopefully, you have a better understanding of indirect ELISA and why it's used. It’s basically just a variation on the original method that allows researchers to get more information from their samples by using different kinds of antibodies. The process is still pretty much the same as direct ELISA, but instead of having just one type of antibody for each sample type, you now have two or more types. This gives you more choices when deciding what kind of detection method would work best with your sample!