applications in biologic screening
TTP Labtech’s mirrorball® is a valuable screening tool for all stages of the antibody discovery process providing high-throughput, high sensitivity, antibody screening for primary screening, clone selection and affinity maturation.
case studies in target-based screening for biologics
High throughput B-cell cloning and hybridoma screening – UCB Pharma case study:
- Tickle, S. et al from UCB Pharma describe the successful design, construction and commissioning of a bespoke automated screening platform, incorporating mirrorball, and how it can be used for screen antibodies against two specific targets. This automated platform can combine high throughput B-cell culture screening with the identification and isolation of single, antigen-specific IgG-secreting B-cells through a proprietary technique called ‘fluorescent foci’. More than 1 billion immune B-cells can now be screened to provide a useful starting point from which to identify the rare therapeutic antibody
Efficient cell and bead multiplexing – MedImmune case study:
- England, E. et al from MedImmune have evaluated a selection of typical cell- and bead-based assays that demonstrate how the multilaser capability of the mirrorball can be exploited to develop highly sensitive multiplex assays. This allows for identifying antibody cross-reactivity and selectivity but significantly without the loss of throughput. In this study the multiplexing of two antibody–cell binding assays (using different dyes) and three cytokine quantitation bead assays (using the same dye at different intensities) was developed. The results demonstrated an improvement of the sensitivity and efficiency of biologics screening
Improving hit quality in biologics screening:
- False positive hits where “good” binders only bind to dead cells can present a problem for traditional assays screening for antibody binding to extracellular targets. This risk is removed when using mirrorball simply by multiplexing antibody binding, total cell number and a dead cell stain. The dead cell stain allows gating such that only antibody binding to live cells is reported in the assay output.
Hybridoma screening process efficiencies:
- In collaboration with a partner, a small-scale study was carried out looking at the screening of His-tagged antibody fragments against cells expressing a target protein. The antibody fragments were supplied in both crude periplasmic preparations (at unknown concentration) and in purified form (at known concentration). The partner company had previously identified and characterised these as “high” and “low” binders encompassing their “best” and “worst” examples of hit molecules. mirrorball correctly identified positive and negative antibody binding fragment activity, even in samples provided in crude periplasmic extract. mirrorball’s one hour no-wash protocol offers a significant process advantage over a multi-stage, multi-wash cell ELISA approach. Additionally, there is no need to purify the antibody fragments prior to screening.
More hits from phage display screening:
- A collaborator was using a phage expression/screening system to identify peptides which bind to cells expressing a target protein. mirrorball screened a small panel of samples for each of two different projects. For project 1, the negative binders were in agreement with previous results. However, in project 2, some phage that were negative by a non-cellular screen against one domain of the target protein, were positive by mirrorball. The fact that mirrorball found more hits in project 2 was likely due to the use of cells expressing the whole target protein in its native conformation. Here mirrorball offered the advantage of identifying hit peptides that have risked being discarded in non-cellular screens.
case study for biologic binding characterisation
Kinetic characterisation of peptide binding in cells:
- In one study biotinylated versions of hit peptides from phage screening were synthesised to further investigate their binding properties to cells expressing the target protein. By re-reading these samples in a no-wash assay format over time mirrorball produced a timecourse of peptide binding which gives an idea of the relative binding affinity. The binding profiles from mirrorball were in agreement with binding affinities determined by other methods. Additionally, peptides synthesised with fluorescent tags facilitated determination of KD and BMAX values. Because mirrorball can analyse adherent cells in situ, this allowed for an easy process with minimal disruption to the cells hence yielding more physiologically-relevant results.
application to ELISA assays
Process optimisation for sandwich ELISA:
- Measurement of such soluble proteins as cytokine or hormone has traditionally been performed using techniques such as sandwich ELISA assays. Although very sensitive, sandwich ELISA protocols have many wash and incubation steps with no possibility of multiplexing. Together, these factors cause a throughput bottleneck. In-house studies successfully transferred a standard colorimetric plate-based off-the-shelf sandwich ELISA kit for IL8 (R&D Systems cat# DY208) to a bead-based, no-wash assay format on mirrorball. This represents a huge process improvement as assay incubations were reduced from 4h 40min, plus additional wash steps in the ELISA, down to one 3 hour incubation with no washes required on the mirrorball.
phenotypic assay applications
In addition to identifying biological molecules binding to a desired target protein on a cell, mirrorball also has the capability to measure the resulting functional phenotypic effects. mirrorball facilitates many different phenotypic applications including cell cycle, apoptosis, proliferation and cytotoxicity.
These results demonstrate that mirrorball is suitable for use in very different stages of biologics screening, and for different assay types.