Nanobodies and Antibodies for Duplexed EGFR/HER2 Immunoassays Using Terbium-to-Quantum Dot FRET

Abstract

Biosensors based on the combination of semiconductor quantum dots (QDs) and Forster resonance energy transfer (FRET) have demonstrated many advantages for simple, fast, sensitive, and multiplexed diagnostics. However, the implementation of QDs as functional standard materials into homogeneous (single-step) FRET immunoassays has not yet been accomplished, because profound investigations of antibody-conjugation strategies concerning their influence on diagnostic performance for quantifying clinical biomarkers are lacking. Here, we report about a systematic study of size, type, orientation, specificity, nonspecific binding, and cross-reactivity of antibodies conjugated to QDs for single and duplexed EGFR and HER2 immunoassays. Time-gated terbium-to-quantum dot FRET detection on a clinical immunoassay fluorescence plate reader (KRYPTOR) enabled a direct comparison of matuzumab, cetuximab, trastuzumab, and pertuzumab monoclonal antibodies and EgA1, EgB4, 11A4, and 18A12 V H H nanobodies conjugated to 605 and 650 nm emitting QDs. Detection limits of 2.9 ng/mL EGFR, using cetuximab and matuzumab conjugates, and 8.0 ng/mL HER2, using oriented 11A4 and 18A12 conjugates, demonstrated the capability of detecting concentrations well below the clinical cutoff values. Multiplexed assays could quantify EGFR and HER2 at low nanomolar concentrations from the same sample. Our results show that careful optimization of QD-antibody conjugation is a prerequisite to implementing QDs into applied clinical diagnostics.

Quantum Dot–Based FRET Immunoassay for HER2 Using Ultrasmall Affinity Proteins

“…All detection limits were in a clinically relevant concentration range of soluble HER2 for breast cancer diagnostics (7–3000 ng mL −1 ) and in the case of the ADAPT6‐His 6 ‐QD625 conjugates even below the clinical cutoff level of 15 ng mL −1 HER2 . The ADAPT6‐His 6 ‐QD625 also outperformed QD conjugates based on nanobodies, Fab' fragments, and IgG antibodies that were previously used in similar Tb‐to‐QD FRET immunoassays for HER2 …”

“…Semiconductor quantum dots (QDs) are important optical nanomaterials for immunoassays, and exhibit unique properties for versatile FRET diagnostics . Nanobodies have been conjugated to QDs and applied for both FRET immunoassays and FRET imaging in combination with supramolecular terbium (Tb) complexes and time‐gated (TG) detection . The application and performance of even smaller engineered scaffold proteins in such QD‐FRET immunosensors remains to be demonstrated.…”

A clinical role for Förster resonance energy transfer in molecular diagnostics of disease

“…Such assays have become standard techniques for diagnostics of various biomarkers for cancer, respiratory, pulmonary, cardiovascular, and infectious diseases, endocrinology and metabolic disorders, and prenatal screening and are commercially available under brand names such as KRYPTOR/TRACE (BRAHMS/ThermoFisher), HTRF (Cisbio), or LANCE (PerkinElmer). Recent trends of FRET-based immunoassay development include multiplexing, the use of nanoparticles (NPs) such as quantum dots (QDs), or the implementation of nanobodies or small artificial binding proteins [6,7].…”

“…FRET can be used in all types of cancer biomarkers such as proteins, SNPs, DNAs, miRNAs, tumor cells, and protein-protein interactions. Most single biomarkers have not reached the level of cancer specificity and sensitivity required for routine clinical use, and thus, biomarker discovery is moving to panels of multiple biomarkers [6,29].…”

“…Our group demonstrated a multiplexed lung cancer immunoassay using five tumor markers, which could allow a distinction of small cell (SCLC) from non-small cell (NSCLC) lung carcinoma by FRET from Tb to multiple dyes [29]. Using FRET from Tb to different QDs, we also developed a duplexed EGFR/HER2 immunoassays, which may aid to more specific lung and breast cancer diagnostics [6].…”