HER2) used to handle the assays is not easily obtained. piezoimmunosensor assay was 0.038 nM with a linear operating range of (0.038 C 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 Amezinium methylsulfate significant in that low cost synthetic peptides could be used in a QCM assay, 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. Introduction Therapeutic monoclonal antibodies (MAbs) have been used to treat human disease. Currently more than 20 MAbs such as Bevacizumab (also known as Avastin), Cetuximab (Erbitux), Rituximab (Rituxan) and Trastuzumab (Herceptin) are being used worldwide to treat conditions including cancer, autoimmune diseases, allergy, cardiovascular disease and transplant rejection.1 Around 300 antibodies are undergoing clinical development and 2915 clinical studies involving antibodies are being carried out.2 Herceptin, for instance, the first FDA-approved humanized MAb for human cancer therapy3 exhibits the ability to inhibit the proliferation of breast tumors by specifically binding to the extracellular domain name IV segment4 of HER2(human epidermal growth factor receptor 2) in Amezinium methylsulfate the membrane of breast cancer cells. Herceptin has been widely used for the treatment of HER2 positive breast cancer since its approval in 1998.3,5,6 However, approximately 1 in 20 patients will produce antibodies to human therapeutic antibodies.7 In some human patients, therapeutic antibodies can be rapidly cleared from the body;2 and, as such, will not be of benefit to the patient. Additionally, the therapeutic activity of a MAb is dependent upon its serum half-life. MAbs used to kill tumor cells or inhibit cell growth need to have a long half-life, while those used as immune-modulatory agonists should have a shorter half-life. If the MAb serum half-life is usually too short or Amezinium methylsulfate too long, then the MAb may not be therapeutically efficacious or may produce deleterious effects. The physicians decision-making process is usually often dependent upon clinically established protocols that may not be suited to every patient. As such, physicians will need assays to monitor serum therapeutic antibody levels to determine if antibody dosage is appropriate to elicit a positive therapeutic effect. Typically, immunoassays are used to determine antibody concentrations in human serum. Physicians use the results of such assays to determine how best to treat patients. At present, a rapid, simple, highly sensitive, inexpensive assay is not readily available for the quantification of therapeutic antibodies in biological specimens. To date, enzyme-linked immunosorbent assays (ELISAs) are still the most widely used technique to detect Herceptin in human serum or plasma.8C11 However, there are limitations of the ELISA method for detection of Herceptin in solutions. First, an ELISA is Rabbit polyclonal to AVEN usually costly and time-consuming. Antibodies for completing an ELISA assay are $200C300 per 100 ug per antibody. These are expensive items. ELISAs require multiple steps and the Herceptin antigen (i.e. HER2) used to carry out the assays is not easily obtained. Genentech, the manufacturer of Herceptin, measures the level of Herceptin in patients by using the extracellular domain name of the HER2 receptor as the coating antigen.8 Maple and coworkers used a full-length HER2 protein as the coating antigen since Genentechs antigen was not commercially available.9 Jamieson, et al described a cell-based ELISA for dection of Herceptin; 11 however, cell-based assays are difficult to standardize (i.e. cell growing and plating conditions can vary). Additionally, Herceptin was designed with human IgG1 constant domains12 and is immunologically comparable to normal human antibodies. As such, it can be difficult to distinguish Herceptin from normal serum antibodies by using traditional immunological reagents and assays Therefore, new bioassays are needed to detect therapeutic antibodies in human samples. Quartz crystal microbalances (QCMs) have been recognized as a standard tool to detect biomolecular interactions (e.g. antigen or peptide/antibody interactions) in real-time without using labels (e.g. fluorescent dye or enzyme-conjugated secondary antibodies). Based on our previous work, QCM can be used with Au coated quartz crystal as a transducer to detect antibody-binding events.13C15 Jiang, et al. 16 used phage display to identify peptides (i.e. mimotopes) that could be used of the HER2 receptor to develop a breast cancer vaccine. We modified one of the HER2 mimotopes (QLGPYELWELSH) and used it to develop a piezoimmunosensor assay to detect Herceptin in solutions. The.