Is it necessary to put question marks at the end of intracellular flow assay? Just now that the most exciting reports on cell signaling and phospho-protein detection are coming out? Wasn’t DNA analysis one of the first diagnostic applications in flow?
The answer to the last question is “yes”, but the small molecular stains are replaced by fluorescent antibodies, and these large molecular structures are causing a problem. At the surface of a cell, we are relatively certain to reach a target, and even there the diagnostic quality procedures of an antibody binding assay are difficult. Going intracellular adds a number of problems to this—like denaturing conditions, hindrance of macromolecule entry, and cellular decomposition.
Many companies are offering solutions for intracellular flow with antibodies and many examples are shown, but little quantitative analysis is performed. An example of such an analysis will be discussed in this article.
While immunofluorescence flow cytometry is a relatively new technique, and one that is used for research purposes in a majority of cases, it is also quickly acquiring diagnostic applications in the study of leukemia and lymphoma, solid tumors, and in immunotherapy, thanks to the massive expansion of monoclonal antibody technology. In most of these cases, the physiological target is located on the surface of the cell and reflects a state of differentiation.
Intracellular antigens of interest for cell signaling such as phosphoproteins are, however, functional markers, not markers of differentiation. Precise detection of this type of molecule in cells of interest is necessary for choosing the most appropriate phosphokinase inhibitor drug for the treatment of an individual’s cancer or, more generally, for phosphokinase inhibitor drug discovery.
There is no reference value for phosphoproteins; their expression can vary in a minute under the influence of activators or inhibitors. Moreover, phosphorylated epitopes are often not available for antibody recognition. Strong protein denaturing procedures are necessary to expose the phosphorylation sites. For the purpose of quantitative analysis it is better to study a less cumbersome type of assay before entering the field of the phosphoproteins.