Validation of results via FACS

The Retrogenix cell microarray technology is highly sensitive with a low rate of false positives. Typically, one or two hits will be identified and shown to be reproducible and specific to the test ligand – these will be confirmed as part of our standard screen. Where screening has been performed using a fluorescence read-out, Retrogenix scientists can also use an orthogonal approach to further validate these hits. This is achieved by performing flow cytometry analysis in order to confirm the specific interactions.

Our flow cytometry service routinely follows on from a full study where required. We are frequently able to use materials that are already available to us at the end of the main screen, avoiding the need to produce and ship further samples to our UK labs.

Retrogenix’s flow cytometry screen takes around 2-3 weeks and provides key additional data to support interpretation of the results from a full receptor identification, target deconvolution or off-target screen.

Expression vectors encoding the hits identified by cell microarray screening, along with appropriate control vectors, are used to transfect HEK293 cells. Live transfectants are then incubated with the test ligand and controls (and with any fluorescent detection antibodies that are required). Each set of transfectants is then analysed by flow cytometry and the results interpreted by Retrogenix scientists.

Results of titration experiments with increasing doses of the test antibody (compared with no-ligand or the positive control) showing the curve shift produced by varying degrees of ligand-receptor binding.

Flow cytometry analysis of binding of a test antibody, an isotype-matched negative control antibody and Rituximab biosimilar control to unfixed transfected cells expressing the known primary receptor, the putative off-target (identified by cell microarray screening) and a CD20 control.


Case Study

A single monoclonal test antibody – developed against a key GPCR target – was provided by the study sponsors along with an isotype-matched negative control antibody.

An initial pre-screen was undertaken to determine the levels of background binding of the test antibody to untransfected HEK293 cells as well as its binding to cells over-expressing the known primary receptor. Binding was assessed using a well-validated AlexaFluor647-labelled anti-human IgG Fc detection antibody, followed by imaging for fluorescence. The pre-screen assessed the suitability of the test antibody for onward screening and helped Retrogenix scientists to select the optimal dose to use throughout the study.

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