Affinity Monoliths for Ultrafast Immunoextraction

Abstract

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.

Chromatographic Immunoassays: Strategies and Recent Developments in The Analysis of Drugs and Biological Agents

“…For example, the use of nanomaterials, such as gold or silver nanoparticles, and quantum dots should continue to lead to new or enhanced detection methods and formats for these methods [19,[85][86][87][88][93][94][95][96][97][98][99][100]109]. The utilization of alternative supports, such as affinity monoliths, is also expected to continue [110][111][112][113][114][115][116]. In addition, further research is anticipated in the development of miniaturized chromatographic immunoassays that can be employed in microfluidic devices and portable or disposable devices [19,[26][27][28]30,[82][83][84][85][86][87][88]100,113,116].…”

“…The utilization of alternative supports, such as affinity monoliths, is also expected to continue [110][111][112][113][114][115][116]. In addition, further research is anticipated in the development of miniaturized chromatographic immunoassays that can be employed in microfluidic devices and portable or disposable devices [19,[26][27][28]30,[82][83][84][85][86][87][88]100,113,116]. These develop ments, in turn, should increase the range of applications for which chromatographic immunoassays can be used in areas that include clinical chemistry, pharmaceutical testing, drug development and biomedical research [7,8].…”

Evaluation of photografted charged sites within polymer monoliths in capillary columns using contactless conductivity detection

“…Examples include the immobilisation of protein A, for the separation of immunoglobulins [6][7][8], lectins such as concavalin A, for the extraction of glycoproteins [9], enzymes such as trypsin, for use as microbioreactors in LC-MS proteomic applications [4], and the immobilisation of immunoglobulins [10]. These varied applications of polymeric monolithic stationary phases for affinity chromatography has been the subject of a recent review by Mallik [11] in which the numerous immobilisation strategies which have previously been reported are discussed.…”

“…These varied applications of polymeric monolithic stationary phases for affinity chromatography has been the subject of a recent review by Mallik [11] in which the numerous immobilisation strategies which have previously been reported are discussed. The most common approaches result in covalent attachment of the protein to a monolith and include the epoxy method [12,13], the Schiff base method [7,10,12], the glutaraldehyde method [7,14], the carbonyldiimidazole method [7,10,12], the disuccinimidyl method, the hydrazide method [10], and the cyanogen bromide method.…”

Preparation of an Immunosorbent and its use in the Solid Phase Extraction of Benzodiazepines

“…In addition to basic properties such as chemical and biochemical inertness, good mechanical stability and uniformity in particle size, the solid support should be easily activated to permit antibody attachment, have appropriate pore size to contain the antibodies, and should be hydrophilic in order to avoid any nonspecific interactions. Traditional supports used in high performance immunoaffinity chromatography (HPIAC) and CE; include silica 11,13,14,15 monolithic silica 16 , synthetic polymers 17 and magnetic beads. 18,19 One advantage of using silica in HPIAC is the availability of this support in a form that is both reproducible and that gives good chromatographic efficiency.…”