AN IMMUNOAFFINITY CAPILLARY ELECTROPHORESIS METHOD FOR THE DETERMINATION OF FRATAXIN IN BLOOD

COLLAZO, D; GRELA D; FARAJ, S; FERRARI, A; SANTOS, J; VIZIOLI, NM

Cartagena de Indias

Simposio; 21st Latin-American Symposium on Biotechnology, Biomedical, Biopharmaceutical, and Industrial Applications of Capillary Electrophoresis and Microchip Technology; 2015

The use of affinity ligands bonded to solid supports in capillary electrophoresis methods has recently received much attention, especially for concentration enrichment, isolation or extraction of analytes present in different complex samples. Particularly, the use of antibodies as high affinity ligands provides the possibility of specific analyte detection with increasedsensitivity. In this work, an immunoaffinity capillary electrophoresis (IACE) method using a macroporous polymeric monolithic support is proposed for the specific isolation of frataxin from a blood sample and its subsequent release from the corresponding immobilized antibody, followed by CE separation and quantification. Deficiency of frataxin, an iron-binding protein, leads to a progressive neuromuscular degenerative disease, known as Friedreich?s ataxia.Short monolithic capillary columns were synthesized inside fused-silica capillaries of 150 μm inner diameter (i.d.) by in situ polymerization of diethylene glycol dimethacrylate (DEGDMA) and glycidyl methacrylate (GMA). The polymerization reaction was initiated and maintained on a Co60 ionizing radiation source. The monolith surface was activated to allow binding of monoclonal antibodies against frataxin. Epoxy groups from GMA were opened by acid hydrolysis, and monoclonal antibodies were then covalently attached. Thechemically modified monolithic microcolums were coupled to two pieces of capillary (75 μm i.d. x 10 cm from the inlet to the microcolumn and 21 cm from the microcolumn to the detector) using polytetrafluoroethylene (PTFE) tubing. Once the capillary was conditioned with a background electrolyte (BGE) composed of 25 mmol/L phosphate buffer solution, pH 7.0, the blood sample was hydrodynamically introduced into the capillary for binding of frataxin to the immobilized antibody. Immediately after this step, the entire capillary wasrinsed with BGE to remove salts and compounds not retained and also to precondition the entire column. The elution of the bound frataxin was performed with 0.1 mol/L glycine-hydrochloric acid, pH 2.7, followed by a plug of BGE. Then, the separation voltage was applied and UV absorption detection was performed at 214 nm. As a consequence of their rapid, simple and reproducible fabrication, monolithic microcolumns chemically modified to which specific antibodies are immobilized resulted very useful for the quantification of frataxin in blood by means of IACE. Furthermore, the method proposed in this work offers a promising analytical tool for the diagnosis of Friedreich?s ataxia.