Comparison of ELISA and SOMAmer EGFR levels.

<p>Serum EGFR ECD levels were measured using EGFR SOMAmer assay (y-axis) and ELISA (x-axis). Black diamond data points showed correlation between the two methods and were used for R² calculation. Grey diamond data points showed much reduced EGFR SOMAmer levels and were not used for R² calculation.</p

Detection of drug-unbound EGFR in patents receiving treatment with an EGFR monoclonal antibody.

<p>Detection of drug-unbound EGFR in patents receiving treatment with an EGFR monoclonal antibody.</p

Intra and inter assay variability of EGFR ECD SOMAmer assay.

<p>Inter-assay variability was determined from 20 runs each of control samples with low (∼27 ng/mL), middle (∼53 ng/mL), or high (∼310 ng/mL) concentrations of EGFR. Intra-assay variability was determined by testing each control sample at least 20 times in a single run.</p

EGFR SOMAmer specificity test.

<p>EGFR SOMAmer reagent was incubated with one of the ErbB proteins followed by labeling of bound protein with Alexa fluor 647. After extensive washing, photocleavage dissociates SOMAmer:protein complex from the bound beads, and the complex is separated by polyacrylamide gel electrophoresis under denaturing conditions. Lane 1, pulled down EGFR; Lane 2, EGFR standard; lane 3, pulled down ErbB2; lane 4, ErbB2 standard; lane 5, pulled down ErbB3; lane 6, ErbB3 standard; lane 7, pulled down ErbB4; lane 8, ErbB4 standard, and lane 9, MW size standards The EGFR SOMAmer showed limited cross-reactivity with ErbB family proteins. Relative to EGFR, the SOMAmer “pulled down” limited amounts of ErbB2 (0.4%), ErbB3 (6.9%), and ErbB4 (1.3%).</p

Linear range of the EGFR SOMAmer assay.

<p>The dilution series was prepared by diluting purified EGFR in 5-fold steps from 600 to 2.5 ng/mL. Each dilution was further diluted to 30 fold using SB17T buffer to mimic the serum dilution. The EGFR SOMAmer assay and quantitative PCR (qPCR) were done as described in Materials and Methods. Triplicate runs were performed per each dilution.</p

EGFR SOMamer assay detects cetuximab and panitumumab-unbound fraction of EGFR.

<p>Normal serum samples were incubated with varying amounts of panitumumab [PANEL A] or cetuximab [PANEL B] for 30 min at room temperature. These samples were then diluted 30 fold, followed by EGFR SOMAmer capture and quantitative PCR (qPCR) (gray bars). EGFR ELISA was also performed on drug-treated samples on the same day (white bars). Triplicate samples were tested at each condition. The vertical lines at each bar represent standard deviations among replicates.</p

Spiked-in purified EGFR to serum is accurately detected by the EGFR SOMAmer assay.

<p>Thirty-fold diluted serum samples were appropriately spiked with three different amounts of purified EGFR ECD (final concentration of 30, 150, and 300 ng/mL plus the endogenous serum EGFR ECD). We then performed the EGFR SOMAmer assay followed by the qPCR. The percent recovery is calculated by dividing SOMAmer-measured EGFR by the expected EGFR level (spiked plus endogenous serum EGFR level), multiplied by one hundred. The codes on the X-axis represent sample number and concentration of spiked EGFR. For example, S_1_300 is serum sample #1 with 300 ng/mL of spiked purified EGFR protein. Each sample was measured three times. The error bars represent standard deviations.</p