A presentation by a MedImmune scientists focused on the application of HTRF for the isolation, optimization, and characterization of antibody drugs. Elizabeth England, who is based in MedImmune’s U.K. facility, specifically discussed a case study involving an anti-IL6 antibody.
A number of assays were developed to identify antibodies with different mechanism of action. Antibodies were found that inhibited the binding of IL-6 to IL-6R or that inhibited the recruitment of the accessory protein gp130.
“The real power of the HTRF technology was demonstrated with the use of the epitope competition assay for lead optimization screening. [The assay’s sensitivity made it] possible to discriminate sub-nanomolar affinity antibodies,” said England. “The development of a second-generation epitope competition assay, whereby a higher-affinity antibody is substituted for the original antibody, can be developed quickly. This is the case because there are fewer variables within the HTRF assay that need to be optimized.”
England and several other conference presenters pointed out a limitation of the HTRF technology that is actually common to all sandwich immunoassays (an antibody pair recognizing the same analyte) and is the consequence of a large excess of analyte with respect to the detecting antibodies. This is the so-called “hook effect,” which can be explained as follows.
While the signal of a sandwich assay increases proportionally (and typically in a linear way) to the analyte concentration, a too-high amount will lead to an increasing quantity of analyte bound to only one of the two conjugates, i.e. not generating any signal. As a consequence, the signal will decrease, and the concentration calculated from the signal measured may seem to correspond to a much lower concentration.
“This can mean that determining the affinity of an interaction becomes more complex as it not simply a case of increasing concentrations until saturation is reached,” explained England.
Compatibility of the toolbox reagents (i.e., reagents individually selected by a researcher as opposed to the use of specific kits) also can sometimes be an issue, she said. For example, antihuman Fc detection of a target protein cannot be used when screening human IgGs. A further limitation is the distance required between donor and acceptor to get energy transfer.
“If very large proteins are used, this distance can be such that there is no excitation of the acceptor molecule,” noted England. “There are also potential issues with steric hindrance from anti-tag detection reagents or from inactivating proteins with direct labeling.”
Nevertheless, HTRF is a very useful technology and researchers just need to be aware of these limitations, she said.