Photo courtesy of Krystyn Wukitsch Foran.
We’ve a talked about conjugation and all the different things secondary antibodies can be tagged with. But that’s not the only thing companies do to antibodies.
Post production steps to modify or purify secondary antibodies can have a big effect on their specificity and on decreasing background.
Fabulous Fab Fragments
Fab fragments are secondary antibodies in which the nonvariable portion of the antibody (called Fc) has been cleaved off leaving only the antigen recognition portion of the antibody (see Advansta’s WiKi on Secondary Antibodies for more information).
Two different types of Fab fragments can be generated. Digestion of an antibody with papain generates 3 pieces: the Fc region and two individual Fab fragments. Digestion with the enzyme pepsin creates only 2 pieces: the Fc region and a divalent Fab fragment called F(ab’)2)
Digestion with papain. Photo courtesy of Wikipedia.
Digestion with pepsin. Photo courtesy of Wikipedia.
Fab. Monovalent Fab fragments are tiny (relatively speaking) and can diffuse easier into tissues and cells when doing immunohistochemistry or trying to access an intracellular antigen. They are also useful for blocking or in special circumstances when binding ratios need to be controlled.
F(ab’)2. Some cells in the body, immune cells in particular, contain Fc receptors; proteins that bind to the Fc portion of antibodies. Fc receptors are not choosy – they don’t care if they are binding antibodies generated against an attacking virus (to stimulate an immune response) or whether they are binding to your antibody that you have selective picked to detect an intracellular protein. They all look the same to them, and if a cell has an Fc receptor it will bind to your antibody and take all of the specificity out or your assay.
Similar to their smaller Fab cousins, F(ab’)2 can gain better access in tissues and cells.
Secondary antibodies are polyclonal antibodies. Immunoglobulins are injected into the animal of choice and serum is obtained that contains all antibodies – all classes and subclasses as well as antibodies that are not specific for the injected antigen. To increase specificity, the antibodies are then run through a column that will selectively enrich for the desired antibody. Need an anti–IgG antibody? Run the serum through a column that will pull out IgM, IgD and IgA molecules.
Adsorbed Secondary Antibody
Despite their pronounced specificity glory, secondary antibodies can cross react between species. If you are doing a double-labeling or multiplex experiment, this could cause you problems.
To get around this, antibodies can be adsorbed against proteins (i.e. serum from other species) to eliminate any antibodies that might cross-react. Be warned: although this can result in a highly specific antibody, sometimes adsorption can also titrate out some of the desired antibody and decrease sensitivity.
Goat Anti-Rabbit IgG (H+L) (min X Hu, Ms, Rat)
A secondary antibody raised in goat, specific for the heavy and light chains of rabbit IgG. The antibody has been adsorbed to prevent cross-reactivity to human, mouse and rat antigens.
It’s simple, right?