Her2 ecd

N2’-Deacetyl-N2’-(3-mercapto-1-oxopropyl)-maytansine (Mertansine, DM1 compound) was obtained from Abcam (Cambridge, UK). Freund’s complete adjuvant (FCA) and Freund’s incomplete adjuvant (FIA) were obtained from Sigma–Aldrich (St. Louis, MO, USA). BSA-conjugated DM1 was prepared by Abfrontier (Seoul, Republic of Korea). Protein A and Protein G were purchased from GE Healthcare. T-DM1 (trade name Kadcyla^®) was purchased from Dongwon Pharmaceutical (Daejeon, Republic of Korea) for laboratory research use. Human anti-trastuzumab, a positive control antibody, was purchased from Bio-Rad (Puchheim, Germany). HER2-ECD was obtained from Sino Biological Inc. (Beijing, China). Human anti-Fc-specific antibody conjugated to peroxidase was purchased from Thermo Fisher Scientific (Waltham, MA, USA). Biotin-conjugated Kadcyla and HER2-ECD were prepared by AbFrontier (Seoul, Republic of Korea). Streptavidin-HRP was purchased from BD Pharmingen^TM (San Diego, CA, USA). Carbonate-bicarbonate buffer, TMB substrate, and stop buffer were purchased from Sigma–Aldrich (St. Louis, MO, USA). A 1x PBS-T was purchased from Fluka (Buchs, Switzerland). Individual Crl:CD (SD) rat blank sera were obtained from BioChemed (Winchester, VA, USA).

A Biacore T200 and a Biacore 3000 instrument (GE Healthcare) were used for biosensor analysis. The extracellular domain of HER2 (HER2_ECD) (Sino Biological, Beijing, China) was immobilized to 210, 310, and 456 RUs on three different flow cells on a CM5 chip by amine coupling in sodium acetate buffer, pH 4.5. A reference flow cell was created by activation and deactivation. On a second CM-5 chip, HSA (Novozymes, Bagsvaerd, Denmark), MSA (Sigma-Aldrich, St. Louis, MO, USA), and BSA (Merck Millipore) were immobilized in the same way. The final immobilization levels were 869, 584, and 779 RUs, respectively. HBS-EP (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% Tween 20, pH 7.4) was used as running buffer and for dilution of the analytes. All experiments were performed at 25 °C with a flow rate of 50 μL/min. The chips were regenerated by injection of 15 mM HCl for 30 s. The binding kinetics was analyzed by the Biacore evaluation software using the one-to-one kinetics model.

Recombinant human EGFR ECD, EGFR ICD, HER2 ECD, HER3 ECD, and HER4 ECD were obtained from Sino Biological Inc. (Beijing, China). They were labelled with a Monolith NT Protein Labelling Kit RED-NHS (Nanotemper, Munich, Germany) and diluted to 250 nM with PBS. Label-free DPBA was diluted at half-concentrations with PBS (100,000–12.21 nM). Label-free EGF was diluted at half-concentrations with PBS (8000–2 nM). Protein samples were mixed with DPBA or EGF and incubated at RT for 5 min. In a competitive assay, EGFR was incubated with DPBA (100 μM) for 60 min at RT before EGF treatment (8000–2 nM). The mixtures were centrifuged at 16,000 × g for 5 min and loaded into capillaries. Microscale thermophoresis measurements were performed in a Monolith NT.115 (Nanotemper, Munich, Germany).