Simple, Rapid Ligand-Binding Assay Using Immobilized Cellular Membranes

Paula Denney Eason, Renée C. Benson, Jenny T. Ly, Rachel A. Saxer, James L. Wilbur, George B. Sigal, Eli N. Glezer, Hans A. Biebuyck, Jacob N. Wohlstadter, and Robert M. Umek

A division of Meso Scale Diagnostics,TM LLC. Simple, Rapid Ligand-Binding Assay Using Immobilized Cellular Membranes

Abstract

This poster presents a robust, receptor-ligand binding assay based upon a novel assay platform developed by Meso Scale DiscoveryTM (MSDTM). MSD’s platform combines array technologies and electrochemiluminescence detection to achieve ultra-fast, highly sensitive assays in a homogeneous format. Cellular membranes containing the EGF receptor were passively adsorbed to MSD proprietary coated electrodes embedded in multi-well plates. Binding of EGF to the EGF receptor was detected by inducing and measuring electrochemiluminescence from a labeled EGF ligand. Approximately 1000 cell equivalents per well yielded a signal to background ratio of 20. The observed KD agrees with that reported in the literature and demonstrates that immobilization of the membranes and modification of the ligand do not alter the binding affinity. Binding specificity was confirmed with two inhibitors. The assay can be readily adapted to facilitate analysis of a broad array of receptor-ligand interactions.

A division of Meso Scale Diagnostics,TM LLC. Simple, Rapid Ligand-Binding Assay Using Immobilized Cellular Membranes

TM Multi-Array TechnologyTM Multi-Array Technology

Unified technology platform with instruments, plates and reagents for drug discovery for drug discovery. Combines the power of microarrays with the sensitivity of electrochemiluminescence Combines the power of microarrays with the sensitivity of electrochemiluminescence.96-, 384- and 1536 microplate formats

96-,Multi-Spot 384- TMand plates 1536 with microplate high density formats. arrays for multiplexing

Multi-SpotSector HTSTMTM platesInstrument: with highHigh densityresolution arrays imaging for multiplexing. detection and robotic integration for HTS and large-scale proteomics TM Sector HTS Instrument: High resolution imaging detection and Sector HTS roboticSector PRintegrationTM Instrument: for HTSMedium and throughputlarge-scale proteomics.benchtop reader for assay development, cellular and molecular biology, research in therapeutic areas, secondary screening, QC. Assays developed on Sector PRTM portInstrument: to Sector Medium HTS. throughput benchtop reader for assay development, cellular and molecular biology, research in therapeutic areas, secondary screening, QC. Assays developed on Sector PR port to Sector HTS.

Sector PR

A division of Meso Scale Diagnostics,TM LLC. Simple, Rapid Ligand-Binding Assay Using Immobilized Cellular Membranes

EGF-EGFR Assay Format

Extra-Cellular Domain Trans-Membrane Domain EGFR A-431 Cells Cytoplasmic Domain

1) Lyse cells 2) Purify membrane fragments 3) Passively adsorb membrane fragments on electrode, 30min MSD Proprietary Coated Carbon Electrodes

Bind labeled EGF (STAG-EGF), 1h STAG-EGF

1)Wash away unbound STAG-EGF 1)Add TPA 2)Add TPA 2)Induce and Measure 3)Induce and Measure electrochemiluminescence (ECL) electrochemiluminescence(ECL)

ECL ECL ECL ECL

Washed Non-washed Format Format

A division of Meso Scale Diagnostics,TM LLC. Simple, Rapid Ligand-Binding Assay Using Immobilized Cellular Membranes

Direct Immobilization of Biologically Active Membranes on Carbon Electrodes

10µg 10µg Total Total Supernatant Supernatant 1 2 1 2 A-431 K-562 Western Silver Quantification of Membrane Immobilization Blot Stain on the Electrode Surface Ten micrograms of total membrane protein was loaded directly onto the gel. For comparison, the supernatant containing the unbound fraction recovered after 10µg total membrane protein was incubated on the electrode band used to quantify for 30 min is shown. α-EGFR antibody was used for the western blot.

4000 Optimal Mass Immobilized on the Electrode Titration of total membrane protein to determine optimal plating 3000 A431 mass. In this 96-well washed format, membranes were immobilized K562 onto MSD proprietary coated carbon electrodes in 5µL/well for 1 hour. 2000 Next, labeled ligand was added in 3% BSA for 1 hour. Unbound

Average Signal ligand was washed, followed by the addition of assay buffer just prior 1000 to electrochemiluminescence.

0 01234 Total Membrane Protein (µg/well)

A division of Meso Scale Diagnostics,TM LLC. Simple, Rapid Ligand-Binding Assay Using Immobilized Cellular Membranes

Binding Characteristics of Membranes are Conserved After Immobilization on Carbon Electrodes

Saturation-Binding Curve Saturation-Binding Curve 96 - Well Washed Format 384 - Well Washed Format 12500 1000

10000 A431 750 A431 K562 K562 7500 500 5000 Average Signal Average Signal KD = 4.7 KD = 9.9 250 2500

0 0 0 50 100 150 200 250 300 0 100 200 300 STAG-EGF (nM) STAG-EGF (nM)

EGF was purchased (Sigma) and labeled with a sulfonated derivative of Ruthenium (II) tris-bipyridine (the electrochemiluminescent label, here abbreviated as STAG). STAG-EGF was purified by FPLC and the molecular weight of the labeled ligand was confirmed by mass-spectral analysis. Purified STAG-EGF was quantified by both a BCA protein assay and ruthenium absorbance at 455nm. As little as 1000 or 300 cell equivalents/well in the 96- and 384-well formats, respectively, produced maximum signal to background. The assay was performed using the standard protocol. The observed KD values in both the 96- and 384-well assays were within 3-5 fold of that reported in scientific literature, demonstrating a very modest change in binding affinity of STAG-EGF relative to native EGF.

A division of Meso Scale Diagnostics,TM LLC. Simple, Rapid Ligand-Binding Assay Using Immobilized Cellular Membranes

Receptors in Immobilized Membranes Retain Sensitivity to Known Inhibitors

Suramin Competition Cold EGF Competition 96-Well Washed Format 96-Well Washed Format

10000 4000 A431 A431 K562 K562 7500 3000

5000 IC50 = 20nM 2000 IC50 = 1.8mM

Average Signal STAG-EGF = 120nM Average Signal STAG-EGF = 15nM 2500 1000

0 0 1e -6 1e -5 1e -4 1e -3 .01 0.1 1 10 0.001 0.01 0.1 1 10 100 EGF (nM) Suramin (mM)

Receptor binding to STAG-EGF was assayed as a function of varying concentrations of unlabeled EGF. The observed IC50 value of the unmodified ligand relative to the STAG-EGF challenge is in agreement with the slightly greater KD observed for the STAG-EGF. Suramin, another compound known to inhibit binding of several growth factor receptors, also inhibited STAG-EGF binding in the assay. The assay was conducted according to the standard protocol except that the labeled ligand and the inhibitor were incubated simultaneously in the plate.

A division of Meso Scale Diagnostics,TM LLC. Simple, Rapid Ligand-Binding Assay Using Immobilized Cellular Membranes

Membranes Immobilized on Carbon Electrodes are Compatible with HTS

384-Well Washed Format 60nM STAG-EGF A431 K562

Z -factors for all 5 plates >0.5 100

Signal Signal to Background = 6.3 A-431 CV = 10.2% K-562 CV = 7.3% CV for all 5 plates = 8.7%

plate 1 plate 2 plate 3 plate 4 plate 5 0.57 0.56 0.63 0.53 0.68 Z -factor 10 Well Number

In this 384-well, washed format, membranes were deposited with a PixSysTM instrument (Cartesian Technologies) and R allowed to incubate on carbon electrodes for 30min. Labeled ligand, 60nM STAG-EGF, in 3% BSA was added by a RapidPlate instrument (Zymark) and incubated for 1 hour at ambient temperature. Unbound ligand was washed away, followed by the addition of assay buffer on the Rapid-Plate just prior to inducing electrochemiluminescence. The assay is adaptable to a fully integrated work system for high-throughput screens.

A division of Meso Scale Diagnostics,TM LLC. Simple, Rapid Ligand-Binding Assay Using Immobilized Cellular Membranes

Conclusion

1. We have developed a simple, rapid assay to detect the binding of the EGF ligand to EGFR via passive adsorption of biologically active membranes onto disposable, MSD proprietary coated carbon electrodes. 2. Quantitative membrane immobilization demonstrates biologically active A-431 and K-562 membranes. As little as 0.5µg (1000 cell equivalents) and 0.15µg (300 cell equivalents) total membrane protein/well, produced maximum signal to background in the 96- and 384-well assays, respectively. 3. Immobilization of the membranes onto MSD proprietary coated carbon electrodes and modification of the ligand result in a very modest alteration in binding affinity.

Observed KDs in both the washed and non-washed formats agree with that reported in literature, while characteristic competition curves with cold ligand and a known inhibitor demonstrate receptor-ligand binding specificity. 4. The assay is adaptable to completely automated 96- and 384-well formats for HTS. A 5-plate screen produced Z-factors > 0.5/plate and interplate CV’s = 8.7% in a 384-well washed format.

A division of Meso Scale Diagnostics,TM LLC.