Hit validation & follow-on analysis

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 at the confirmation screen stage. Where screening has been performed using a fluorescence read-out, Retrogenix scientists can now investigate these hits further by performing follow-on flow cytometry analysis in order to validate the specific interactions.

Our flow cytometry service routinely follows on from a full study. This allows clients to opt into the service dependent on results rather than commit upfront. 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.

facs

Follow-on 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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Pfizer
The University of Sheffield
Aveo Oncology - The Human Response
Theraclone Sciences
BioInvent
AstraZeneca
Bluebird Bio
The Center for Infectious Disease Research
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The University of Copenhagen
Lund University
MedImmune
NIH - National Institutes of Health
The University of Pennsylvania
Scripps Florida - The Scripps Research Institute
Peptinnovate Ltd - Unlocking Nature's Potential
Working with Retrogenix was a breeze. We had great communication with the Retrogenix team along the entire process of our protein screen and they always delivered. Their membrane protein library is a great tool for discovering novel cell surface interaction partners.
Dr. Joseph Smith, Center for Infectious Disease Research, Seattle, WA