ABSTRACT
Biopharmaceuticals administered to the human body have the potential to trigger the production of anti-drug (also called anti-therapeutic) antibodies (ADA) that can neutralize the therapeutic activity. For antibody therapeutics, cell-based neutralizing ADA assays are frequently used to evaluate ADA in clinical studies. We developed a method to detect neutralizing antibodies against MEDI-575, a fully human IgG2κ antagonistic antibody against PDGFR-α. We evaluated three assay formats, two of which measured late responses, cell proliferation and apoptosis, whereas the third assay detected an early signaling event, phosphorylation of PDGFR-α. Measuring phosphorylation provided a superior assay window and therefore was developed as a neutralizing ADA (NAb) assay. Matrix interference, however, was significant, and could be identified to be caused by PDGF-AA and PDGF-AB, apparently the two most abundant ligands of PDGFR-α present in human serum samples. A simple pre-treatment step, addition of an inhibitory antibody to PDGF-A, a subunit present in PDGF-AA and PDGF-AB, was found to eliminate matrix interference, increasing assay reliability and sensitivity. We integrated the pre-treatment step into assay development and qualified a robust NAb assay.
Subject(s)
Antibodies, Monoclonal/immunology , Antibodies, Neutralizing/blood , Immunoassay/methods , Receptor, Platelet-Derived Growth Factor alpha/antagonists & inhibitors , Antibodies, Monoclonal/adverse effects , Apoptosis/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival/drug effects , Dose-Response Relationship, Drug , Humans , Ligands , Phosphorylation , Platelet-Derived Growth Factor/immunology , Platelet-Derived Growth Factor/metabolism , Predictive Value of Tests , Receptor, Platelet-Derived Growth Factor alpha/immunology , Receptor, Platelet-Derived Growth Factor alpha/metabolism , Reproducibility of Results , Signal Transduction/drug effectsABSTRACT
A separation concept, selective ion extraction (SIE), is proposed on the basis of the combination of hydrodynamic and electrokinetic flow controls in microfluidic devices. Using a control system with multiple pressure and voltage sources, the hydrodynamic flow and electric field in any section of the microfluidic network can be set to desired values. Mixtures of compounds sent into a T-junction on a chip can be completely separated into different channels on the basis of their electrophoretic mobilities. A simple velocity balance model proved useful for predicting the voltage and pressure settings needed for separation. SIE provides a highly efficient separation with minimal additional dispersion. It is an ideal technique for high-throughput screening systems and demonstrates the power of lab-on-a-chip systems.
