By Levi Clancy for Student Reader on
There are two types of antibody staining: direct staining and indirect staining. Direct staining is faster but costlier than indirect staining.
Direct staining uses a monoclonal antibody that has been previously conjugated to a fluorochrome (ie, FITC or PE). The cells are simply incubated at 4°C for a half-hour with the antibody, then washed several times to remove weakly or nonspecifically bound antibodies. The cells are then ready for microscopy or flow cytometry.
Indrect staining uses a monoclonal antibody with no bound label. The sample is incubated with this primary antibody, then washed, then the sample is incubated with a secondary antibody. The secondary, fluorescent antibody binds the general class of the unlabeled primary antibody.
Antibody quality and titer should be determined for every new antibody, and is recommended for new batches of previously tested antibodies.
Generally, antibodies to non-human mammals are of poorer quality than those that bind human cells, due to a lack of regulatory rules governing their production. Properly titering an antibody will quantify its quality. It is imperative to not use too little antibody that not enough cells of interest are bound. And it is crucial to not saturate your sample with so much antibody that the antibodies bind promiscuously.
Since 3µg per 100µL (106 cells) is the initial concentration where the nonspecific binding component of all IgGs becomes detectable, this is a convenient starting concentration for titering.
Regardless of how specific is your antibody, its Fc end will promiscuously bind any cells with Fc receptors; and dead cells are notorious for soaking up antibodies.
Thus it is imperative to use an isotype control as a negative control for your antibody. An exception is with routine immuno-phenotyping where positive staining may be distinguishably bright enough. An isotype control is of the same immunoglobulin isotype (subclass) as the experimental antibody. It will allow you to ascertain how much background stain is due to irrelevant stickiness such as dead cells and Fc receptors. This isotype control will be as similar as possible to your experimental antibody but have a different specificity.
For example, consider if you stained your human cells with 10µg/mL of a murine IgG2a antibody (conjugated with six fluorescein molecules per antibody) that is specific to a T lymphocyte CD3 protein. An appropriate isotype control would be another murine IgG2a antibody, at the same concentration, with the same fluorescein conjugation ratio, but with a specificity for any antigen unlikely to be found on a human blood cell.