Subproject #1 Analysis of biological samples using Immunoaffinity capillary electrophoresis (ICE) Capillary electrophoresis combined with lased induced fluorescent (LIF) detection is an established method for analyzing biological samples. The small sample volumes, short analysis times and low reagent use are advantageous over more traditional methods such as HPLC. Derivitization of the capillary with antibodies allows for specific capture and detection of single or multiple analytes from a complex biological sample. ICE increases the limits of detection for CE without the need for pre-processing samples. Additionally this technique can be used on both a traditional CE machine as well as a micro-chip CE. ICE has been used to process samples from several collaborators on the NIH campus. Studies include the following: -Analysis of human plasma samples for cytokines indicated in kidney diseases -Tissue analysis from mice and rats from disease model studies looking at Alzheimers -Analysis of plasma samples for antigens indicated in HIV disease models -Analysis of tissue samples from rats given cytokine inhibition drugs Subproject #2 Multiplex ELSIA analysis of cell secretions Multiplex ELISA improves upon the traditional ELISA format. Rather than detecting a single antigen in a single sample, multiplex technology allows for the detection of up to 16 antigens from a single sample. This reduces the overall time and cost associated with ELISA and requires far less sample volume to produce more information. The multiplex format is customizable to allow researchers to investigate a unique array of proteins and peptides. Studies using multiplex ELISA include: -Analysis of cell secretion products from astrocytes stimulated with growth factors -Analysis of cell secretion products from macrophage stimulated with growth factors Subproject #3 Surface Plasmon Resonance Biacore 3000 detection system uses Surface Plasmon Resonance (SPR) to monitor the refractive index (RI) change as molecules interact at the sensor chip surface. SPR is the excitation of surface plasmons by light. The ligand is immobilized on the sensor chip surface, while the analyte is injected in continuous flow over the surface. If an interaction takes place, the surface concentration of the analyte is increased which results in a RI change. SPR technology enables label-free real-time detection and monitoring of biomolecular events and provides qualitative and quantitative information on specificity of binding between two molecules, affinity, and kinetics of the interaction. Experimental design is highly flexible and the technology can be applied to protein interaction with other proteins, nucleic acids, lipids, small molecules. Studies using surface Plasmon resonance include: -Immobilization of Neutravidin (NA), strep-tadded Adenilate Cyclase (CyA) capturing and binding of cAMP Receptor (CRP) protein to CyA -Immobilization of 1D4 antibodies, Rho-tagged peripheral cannabinoid receptor CB2 capturing and the binding of antibody fragments (nanobodies, NBs) to CB2
