An antibody discovery scientist is developing a fluorescence cytometry assay to
ID: 3479772 • Letter: A
Question
An antibody discovery scientist is developing a fluorescence cytometry assay to screen murine antibodies for binding a cell surface target receptor, using a fluorescently labeled anti-mIgG secondary antibody. To reduce the number of wash steps in the assay and improve throughput, she tests an “all-in-one” assay, in which the target cells are mixed together with both primary and secondary antibodies. To her surprise, several antibodies show much (~1000-fold) lower EC50 values in this assay compared to an assay where cells are incubated with primary antibody, then washed before applying the fluorescent secondary antibody. What could be the reason for the apparently better affinity measured in the “all-in-one” assay? (HINT: both the primary and secondary antibodies are bivalent)
Explanation / Answer
EC50 value is the concentration of the antibody inducing a binding response halfway between the baseline and maximum after a specific period of exposure. It is the concentration where the half antibody amount has bound to the antigens.
In bivalent antibodies, two or more binding sites can cross link one antigen to another. Hence, bivalent antigens bind to two antigenic epitopes. Monoclonal antibodies are usually monovalent while polyclonal antibodies are bivalent or multivalent.
Secondary antibodies are usually bivalent where the two Fab region that can bind the Fc portion of two primary antibodies. The Fc region of secondary antibody is bound to the fluorescence tags. This bivalent epitope binding helps to enhance signals. When both bivalent primary and secondary antibodies are added to the cell for fluorescence cytometry, the primary antibody can bind to two different epitopes of the cell surface target receptor. There may be non-specificity if any one of the receptor shares homology with any other antigenic site on other proteins.
The secondary antibody is mouse IgG type. It can coat the antigens in circulation and speed antigen uptake. The secondary anti-mIgG is raised against heavy and light chain of mouse IgG. Hence, when primary and secondary antibodies are added together, the secondary antibody autoreact/bind with the epitopes on the primary antibody. Further, the primary antibody will also bind the specific antigens of cell surface receptor, which is again bound to Fab of secondary antibody to generate a signal. Hence, a huge reaction complex be formed, thereby giving lower EC50 values and higher affinities.
If both antibodies are added in two steps, primary antibody will first bind the specific antigens of the cell surface receptor. Washing will remove excess antigens due to washing of target cells. When the secondary antibody is added in the second step, this antibody will only have Fc portion on the secondary receptor, thereby giving a specific signal. This results in higher EC50 values as more primary antibodies are required for detection.
It should be noted that the lower EC50 values does not indicate higher specificity of the antibodies. In the all in on protocol, since secondary antibodies can bind to epitopes on primary antibodies, non-specific precipitates will form. The assay only measures fluorescence intensity of the signal and links it to affinity. It does not indicate specificity. If some antibodies are better in binding to the target antigens, they will show very low EC50 values.