Generally speaking, there are only two methods employed for identifying antigens with a fluorescent dye; the first involves labeling (conjugating) the primary antibody and is known as the ‘direct’ method, while the second involves labeling the secondary antibody and is known as the ‘indirect’ method. It should be noted that the indirect method nearly always produces a brighter signal and often identifies additional target antigens than the direct method does (assuming the same concentration/incubation conditions), due to the amplification effect described below.
Although the 2-dimensional diagrams shown above attempt to demonstrate a relationship between the quantity of bound antibody molecules and the degree of ‘staining’ (i.e. signal strength), the actual quantity of secondary antibodies that bind to primary antibodies - as well as the quantity of ‘label’ molecules that are conjugated to the secondary antibody - is difficult to quantify.
The diagram at left is intended to demonstrate the actual, 3-dimensional quality of the binding between primary and secondary antibodies and the amplification effect that is created in this manner.
Written by Joseph Myers#2020#Blog#IHCandMolecular
Interested in learning more about IHC? Check out Joe Myers' lectures in the NSH QIHC Prep Course, available on elearn.nsh.org.