ELISA assay employing epitope-specific monoclonal antibodies to quantify circulating HER2 with potential application in monitoring cancer patients undergoing therapy with trastuzumab

Abstract

Circulating HER2 extracellular domain (HER2 ECD) levels were proposed as a surrogate for HER2 tissue expression to monitor breast cancer patients for early relapse or responses to standard or HER2-targeted therapies, such as the monoclonal antibody (mAb) trastuzumab. Currently, available commercial ELISA assays for HER2 ECD rely on antibodies recognizing undisclosed or unknown epitopes. In this work, two ELISA assays employing MGR2 and MGR3 epitope-specific mAbs for HER2 ECD were developed and validated, showing good assay precision and linearity of the dose-response signal within the dynamic range of 0.19-12.50 ng mL^-1 and detection limits of 0.76 and 0.75 ng mL^-1 for the MGR2 and MGR3 assays, respectively. The developed assay showed a good agreement with two widely used commercial kits for HER2 ECD quantification in serum samples from breast cancer patients. A complete characterization of mAb-HER2 ECD interaction was performed by means of surface plasmon resonance using trastuzumab as control for both epitope mapping and kinetics analysis. The epitopes recognized by the two mAbs showed no overlap with trastuzumab, which was confirmed by trastuzumab interference analysis in serum samples. The method showed to be a practical approach to determine HER2 ECD with a high degree of sensitivity, reliability and recovery in samples containing mAbs-based therapies.

Figure 1

Competitive binding test for mAbs MGR2, MGR3, and trastuzumab toward immobilized HER2 ECD, determined by Biacore X100. (a) Trastuzumab (1st injection) vs MGR3 (2nd injection); (b) MGR3 (1st injection) vs trastuzumab (2nd injection); (c) trastuzumab (1st injection) vs MGR2 (2nd injection); (d) MGR2 (1st injection) vs trastuzumab (2nd injection); (e) MGR3 (1st injection) vs MGR2 (2nd injection) (f) MGR2 (1st injection) vs MGR3 (2nd injection).

Figure 2

SPR sensograms showing the kinetic analysis performed on HER2 ECD antigen immobilized on CM5 chip. mAbs were tested at the following concentrations: (a) 0.25–30 nM for trastuzumab; (b) 1–75 nM for MGR2; (c) 2.5–200 nM for MGR3. Association time 500 s, dissociation time 400 s.

Figure 3

ELISA standard curve for HER2 ECD quantification in the range 0–12.5 ng mL⁻¹, employing (a) 2 µg mL⁻¹ MGR2 or (b) 5 µg mL⁻¹ MGR3 mAbs as the capture antibodies and anti-HER2 ECD pAb as detection antibody. Error bars indicate standard deviation (SD).

Figure 4

ELISA standard curve for HER2 ECD quantification in the range 0.19–12.5 ng mL⁻¹, employing 5 µg mL⁻¹ MGR3 as the capture antibody and 15 µg mL⁻¹ biotinylated MGR2 as detector mAb. Error bars indicate SD.

Figure 5

Bland-Altman plot of differences between (a) ADVIA Centaur or (b) Quantikine HER2 ELISA kit and in house developed ELISA. Passing-Bablok regression analysis (n = 11 serum samples) comparing (c) ADVIA Centaur or (d) Quantikine HER2 ELISA kit with the in-house ELISA. In Bland-Altman analysis (panels a and b), the difference between the tested methods is plotted against the mean for HER2 ECD level. Black dotted lines show limits of agreement (95% CI) and the blue line shows the mean value of the differences between the two methods under comparison (the bias). The red dotted line is the zero line for assessing the discrepancy of the observed mean difference from zero. In Passing–Bablok agreement analyses (panels c and d), the data obtained with the in-house ELISA (y axis) is plotted against those of the commercial reference methods (x axis) together with the identity line (y = x) and the regression line. The regression line is continuous and blue, the red dotted line is the identity line (y = x) and the black dashed lines are the CI. Results are summarised as it follows. (c) regression equation, y = 1.41 + 0.87x; intercept, 1.41 (95% CI −5.85 to 7.39); slope, 0.87 (95% CI 0.33 to 1.56). (d) regression equation, y = −0.37 + 1.08x; intercept, −0.37 (95% CI −3.17 to 2.86); slope, 1.08 (95% CI 0.72 to 1.40).

Figure 6

Determination of HER2 ECD only (black dots) or pre-incubated with trastuzumab at 50 (red triangles) and 200 (blue squares) µg mL⁻¹ employing (a) MGR2- or (b) MGR3-based assay and (c) the dual mAbs sandwich ELISA with MGR3 in coating and biotinylated MGR2 as the detection antibody. Error bars indicate SD but are too small to be visible.