No abstract

Therapeutic antibodies are antigenically similar to human antibodies and are difficult to detect in assays of human serum samples without the use of the therapeutic antibody’s complementary antigen. Herein for the first time we established a platform to detect Herceptin in solutions by using a small (< 2.2 kDa), inexpensive, highly stable, HER2 Mimotope-derived synthetic peptide immobilized on the surface of Au quartz electrode. We used the HER2 mimetope as a substitute for the HER2 receptor protein in piezoimmunosensor or quartz crystal microbalance (QCM) assays to detect Herceptin in human serum. We demonstrated that assay sensitivity was dependent upon the amino acids used to tether and link the peptide to the sensor surface, and the buffers used to carry out the assays. The detection limit of the piezoimmunosensor assay was 0.038 nM with a linear operating range of (0.038 – 0.859 nM). Little non-specific binding to other therapeutic antibodies (Avastin and Rituxan) was observed. Levels of Herceptin in serum samples obtained from treated patients, as ascertained using the synthetic peptide-based QCM assay, were typical for those treated with Herceptin. The findings of this study are significant in that low cost synthetic peptides could be used in a QCM assay, in lieu of native or recombinant antigens or capture antibodies, to rapidly detect a therapeutic antibody in human serum. The results suggested that a synthetic peptide bearing a particular functional sequence could be applied for developing a new generation of affinity-based immunosensors to detect a broad range of clinical biomarkers.

CD20, expressed on greater than 90% of B-lymphocytic lymphomas, is an attractive target for antibody therapy. Rituximab is a chimeric murine/human-engineered monoclonal antibody and can selectively deplete CD20-expressing cells in peripheral blood and lymphoid tissues. The immobilization of B-lymphoblast-like Burkitt's lymphoma Raji cells on the quartz crystal microbalance (QCM) gold electrode surface using RGD tripeptide was electrochemically confirmed. The real-time processes of attachment of Raji cells on the gold electrode and the subsequent binding of Rituximab to the cells were studied using QCM biosensor. The interaction between Rituximab and Raji cells led to the increased resonant frequency shifts (Δf0) in the studied antibody concentration range from 5 to 250 µg mL−1 following the Langmuir adsorption model. From these observations, the apparent binding constant between a single-layer of Rituximab and Raji cells was calculated to be 1.6×106 M−1. Control experiments using other therapeutic antibodies (i.e., Trastuzumab and Bevacizumab) and different cells (i.e., T cells and endothelial cells) proved the specific interaction between Rituximab and B cells. The effects of Ca2+ and Mn2+ ions on the Rituximab-Raji cell interaction were also studied providing the enhanced QCM signals, in particular, further indicating that CD20 is a calcium ion channel that can transport these metal ions into the cells and accelerate the cell lysis induced by Rituximab. Thus the real time capability of QCM and its simplicity of operation are highly suitable for multipurpose studies on living cells including cell-immobilization, cytotoxicity of drugs, and the cell action mechanisms.

A label-free and reagent-free peptide mimotope capacitive biosensor has been developed for cancer drug (trastuzumab) quantification based on nonfaradic readout. The low sensitivity issue of capacitive biosensors was overcome with two innovations: peptide mimotope mixed self-assembled monolayer (SAM) biointerface and dilution of the analysis buffer. Signal amplification was achieved through dilution of phosphate-buffered saline (PBS) to tune C to dominate the overall capacitance change upon target binding, which contribution is often negligible without dilution. After 1000× dilution, the limit of detection was lowered 500-fold (0.22 μg/mL) and the sensitivity was increased 20-fold [0.04192 (μg/mL)] in comparison with undiluted PBS. The proposed signal amplification strategy is more straightforward and practical compared to biorecognition element engineering and other strategies. The proposed method was further applied to planar electrodes for optimizing sensing response time to less than 1 min.