A Homogeneous Multiplex Immunoassay for RTK Phosphorylation State that is Adaptable to HTS

Jane Schmidt, Ph.D., Judy Haning, Shaya Anderson, & Richard K. Fuerstenberg

INTRODUCTION

Receptor Tyrosine Kinases (RTKs) are widely expressed transmembrane receptors for growth factors and other extracellular signaling molecules. Binding of a specific ligand leads to phospho-rylation at specific sites in the cytoplasmic tail that triggers further signal transduction processes. RTKs play critical roles in the regulation of developmental processes, including cell survival, proliferation, and motility. In addition, when unregulated, they are involved in cancer formation. For these reasons, RTKs have become popular therapeutic targets for drug development.

The Human RTK Magnetic Luminex® Screening Assays are bead-based multiplex immunoassays for the analysis of RTK phosphorylation. Due to the homogeneous assay format and small sample volume required, these assays can be readily adapted for high-throughput screening with minimal manual intervention from cell treatment to data collection. To demonstrate this, several different cell lines were grown in 96-well plates, treated with various stimulants or inhibitors, and lysed in the same plate. Diluted lysates were transferred to the assay plate for analysis of RTK phosphorylation using this Kit. Using these assays, relative phosphorylation states were successfully determined for the various cell types and treatments.

MATERIALS AND METHODS

Cell Culture and Reagents
A431 human epidermoid carcinoma cells, MDA-MB-453 human breast cancer cells, and CCD1070SK human foreskin fibroblast cells were originally from American Type Culture Collection (Manassas, VA). The cells were maintained in the recommended media. All biochemical reagents were from Tocris, an R&D Systems Company (Minneapolis, MN), including the kinase inhibitors HDS 029 (Catalog # 2646), SU 6668 (Catalog # 3335), and GTP 14564 (Catalog # 2086), and the Insulin Receptor/EGF R activator Demethylasterriquinone (DAQ) B1 (Catalog # 1819). All recombinant proteins were from R&D Systems, including recombinant human EGF (Catalog # 236-EG), NRG1-beta 1/HRG1-beta 1 (Catalog # 396-HB), and PDGF-BB (Catalog # 220-BB).

Phosphorylation was measured using either the Human RTK Luminex Screening Assay Kit A (Catalog Number # VMAPMAGA) or the Human RTK Luminex Screening Assay Kit B (Catalog Number # VMAPMAGB). Data were collected using the Luminex MAGPIX® analyzer.

Phosphorylation and Inhibition of EGF R
A431 cells were grown in a 96-well, high binding tissue culture plate to 95% confluency. For inhibitor experiments, the cells were incubated with 220 nM HDS 029 for 1 hour prior to stimulation. Cells were then stimulated for 5 minutes with 500 μM DAQ B1 to induce EGF R phosphorylation. The cells were then lysed and assayed.

In addition, A431 cells were grown in a 96-well, high binding tissue culture plate to 95% confluency. For inhibitor experiments, the cells were incubated with 110 nM HDS 029 for 1 hour prior to stimulation. The cells were then stimulated for 5 minutes with 100 ng/mL recombinant human EGF to induce tyrosine EGF R phosphorylation. The cells were then lysed and assayed.

Phosphorylation and inhibition of ErbB3
MDA-MB 453 cells were grown in a 96-well, high binding tissue culture plate to 95% confluency. For inhibitor experiments, the cells were incubated with 220 nM HDS 029 for 1 hour prior to stimulation. Cells were stimulated for 5 minutes with 100 ng/mL recombinant human NRG1-beta1/HRG1-beta1 to induce ErbB3 phosphorylation. The cells were then lysed and assayed.

Phosphorylation and inhibition of PDGF Rb
CCD1070SK cells were grown in a 96-well, high binding tissue culture plate to 95% confluency. For inhibitor experiments, the cells were incubated with 5 μM GTP 14564 or 0.3 μM SU 6668 for 1 hour prior to stimulation. The cells were then stimulated for 5 minutes with 100 ng/mL recombinant human PDGF-BB to induce PDGF R beta phosphorylation. The cells were then lysed and assayed.

HOMOGENEOUS MULTIPLEX ASSAY

Assay Principle
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RESULTS

TABLE 1.

Cell Type RTK Sensitivity
(ng lysate/well)
Magnetic Luminex Screening Assay
Hek293-EphA5 MSP R <1 Magnetic Luminex Screening Assay

Human RTK Kit A

TrkA 10
Flk-2/Flt-3 1
EphA4 254
EphA5 1
EphA6 41
EphA7 1
EphA8 508
EphB1 508
EphB2 1015
EphB3 652
EphB4 254
EphB6 5
HGF R 10
HEK293-EPHA1 Ret 1
EphA1 674
EphA2 1428
MDA-MB 453 ErbB2 713
ErbB3 45
A431 EGF R 65
C6-SCF R SCF R 2416
HepG2 ErbB4 3
M-CSF R 3
IGF-I R 178
Insulin R 2 Magnetic Luminex Screening Assay

Human RTK Kit B

EOL-1 FGF R3 24
Tie-1 24
PDGF Ra 12
ROR-1 <1
Mer <1
TrkC 1
MuSK 2
DDR1 6
DDR2 24
PDGF Rb 24
FGF R1 24
TrkB <1
CCK4 1
Dtk 49
ROR2 <1
LTK <1
ALK <1
A172 Tie-2 5
Axl 12
VEGF R3 5
HEK293 FGF4 FGF R4 166
In Vitro*

*Measured in an in vitro binding assay with recombinant human VEGF

VEGF R1 4
VEGF R2 10

Table 1. Sensitivity of Human RTK Magnetic Luminex Screening Assay. Cells were stimulated either by treating with specific ligands or with pervanadate. Cell lysates were serially diluted and run in both Magnetic Luminex Screening Assay RTK A and B assays as 25-plexes. Sensitivity was defined as the minimum total lysate protein per well required to produce a median fluorescence intensity (MFI) of at least 2 times the baseline signal. For RTK Panel A, the sensitivity is typically less than 2500 ng (2.5 μg) of lysate per well; for RTK Panel B, the sensitivity is typically less than 200 ng (0.2 μg) of lysate per well.

RESULTS

EGF R Phosphorylation & Inhibition

EGF R Phosphorylation & Inhibition
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Figure 1. A431 cells were unstimulated, stimulated with the Insulin Receptor/EGF R activator DAQ B1, or pretreated with the ErbB inhibitor HDS 029 followed by stimulation with DAQ B1. Changes in EGF R phosphorylation were measured using the Human RTK Magnetic Luminex Screening Assay.

EGF R Phosphorylation & Inhibition

EGF R Phosphorylation & Inhibition
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Figure 2. A431 cells were unstimulated, stimulated with EGF, or pretreated with the ErbB inhibitor HDS 029 before stimulation with EGF. Changes in EGF R phosphorylation were measured using the VersaMAP Magnetic Custom Human RTK Magnetic Luminex Screening Assay.

ErbB3 Phosphorylation & Inhibition

EGF R Phosphorylation & Inhibition
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Figure 3. MDA-MB 453 human breast cancer cells were unstimulated, stimulated with NRG1-beta 1/HRG1-beta 1, or pretreated with the ErbB inhibitor HDS 029, followed by stimulation with NRG1-beta 1/HRG1-beta 1. Changes in ErbB3 phosphorylation were measured using the Human RTK Luminex Screening Assay.

PDGF R beta Phosphorylation & Inhibition

EGF R Phosphorylation & Inhibition
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Figure 4. CCD1070SK human foreskin fibroblast cells were unstimulated, stimulated with PDGF-BB, pretreated with the Class III RTK inhibitor GTP 14564 or the PDGF R/VEGF R/FGF R inhibitor SU 6668, followed by PDGF-BB stimulation. Changes in PDGF Rb phosphorylation were measured using the Human RTK Magnetic Luminex Screening Assay.

CONCLUSIONS

  • The assay described is a homogeneous multiplex immunoassay that allows cells to be grown in 96-well plates, treated, lysed, and tested in a continuous process that requires no wash steps.
  • This assay offers a small sample size requirement, with typically < 2.5 μg and as little as 1 ng of total cellular protein in a sample volume of 25 μL.
  • When used as a screening tool, this assay can allow for the rapid screening and selection of RTK inhibitors for further analyses.
  • Magnetic LUminex Assays are available as complete ready-to-use kits that allow the user to choose as many as 25 RTKs for primary screening, or as smaller panels for targeted follow-up analysis.

For research use only. Not for use in diagnostic procedures.

NOTE: This product was formerly known as VersaMAPTM Magnetic Multiplex Kits