Affinity monoliths for ultrafast immunoextraction.
Affinity monoliths based on a copolymer of glycidyl methacrylate and ethylene dimethacrylate were developed for ultrafast immunoextractions. Rabbit immunoglobulin G (IgG) and anti-FITC antibodies were used as model ligands for this work. The antibody content of the monoliths was optimized by varying both the polymerization and immobilization conditions for preparing such supports. The temperature and porogen composition used during polymerization showed significant effects on monolith morphology and on the amount of antibodies that could be coupled to these materials. The effects of various immobilization procedures and coupling conditions were also evaluated, including the coupling temperature, pH, protein concentration, and use of high buffer concentrations. The maximum ligand density obtained for rabbit IgG was approximately 60 mg/g. When a 4.5 mm i.d. x 0.95 mm monolith disk containing anti-FITC antibodies was used, 95% extraction of fluorescein was achieved in 100 ms. These properties make such monoliths attractive for work in the rapid isolation of analytes from biological samples. Similar columns can be developed for other targets by varying the types of antibodies or binding agents placed within the monoliths.
Jiang
Tao
T
Department of Chemistry, University of Nebraska-Lincoln, Lincoln, Nebraska 68588-0304, USA.
Mallik
Rangan
R
Hage
David S
DS
D000818:Animals; D000906:Antibodies / Q000737:chemistry; D002846:Chromatography, Affinity / Q000295:instrumentation / Q000379:methods; D004852:Epoxy Compounds / Q000737:chemistry; D004867:Equipment Design; D019793:Fluorescein / Q000302:isolation & purification; D006863:Hydrogen-Ion Concentration; D007120:Immunochemistry / Q000295:instrumentation* / Q000379:methods*; D007074:Immunoglobulin G / Q000276:immunology; D008689:Methacrylates / Q000737:chemistry; D011817:Rabbits; D013696:Temperature
D000906:Antibodies; D004852:Epoxy Compounds; D007074:Immunoglobulin G; D008689:Methacrylates; C004919:ethylene dimethacrylate; C007870:glycidyl methacrylate; D019793:Fluorescein