Detection of trastuzumab (T) level in human serum using quartz crystal microbalance (QCM) piezo-immunosensors by immobilization of a HER2 mimotope-derived synthetic peptide and its potential application in breast cancer.

Abstract

628 Background: Varied clinico-pathological response to monoclonal antibodies like T has been reported either due to presence of antibodies, rapid clearance or low density of target receptor/antigens. This demands need for an assay to monitor serum T therapeutic levels to ensure appropriate dosage. Enzyme-linked immunosorbent assay (ELISA) is still the most widely used technique to detect T level in human serum which is expensive and time consuming. For the first time, we established a platform to detect T level by using a small (<2.2 kDa), inexpensive, highly stable HER2 mimotope-derived synthetic peptide immobilized on the surface of a gold quartz electrode. Methods: HER2 mimotope was used as a substitute for the HER2 receptor protein in QCM assays to detect T level. The validation samples were prepared from the standard T solution in 10% human serum at three concentrations (10, 20 and 40 ug/ml). The changes in frequencies (ΔF) of sera from 3 female patients , 61, 32 and 44 years old , with ER/PR positive, HER2/neu positive metastatic breast cancer were obtained by calculating the differences between frequency shifts in pre and post T infusion.T level was calculated by equation, (ΔF +1.0022) ÷ 0.9997 μg / ml. Results: We showed that assay sensitivity was dependent upon the amino acids used to tether and link the peptide to the sensor surface and the buffers used. QCM assay was capable of detecting T serum level as low as 0.038 nM (linear operating range of 0.038–0.859 nM). T levels of 3 patients were 43.34, 121.96 and 193.18 μg /ml corresponding to pre and post infusion ΔF of 3.33, 11.19 and 18.31 respectively. The time frame of assay was 20-30 minutes. These results were in concordance with previously published results using ELISA. Conclusions: For the first time, we have established a low cost, highly sensitive, fast, synthetic peptide based QCM assay which could be used as a basis for developing a new generation of affinity-based Immunosensor assays to monitor serum levels of T and other monoclonal antibodies, helping physicians to determine the clinical efficacy of these drugs and ensuring appropriate dosages.