title: Sorption of his-tagged Protein G and Protein G onto chitosan/divalent metal ion sorbent used for detection of microcystin-LR.
abstract: A highly sensitive, specific, simple, and rapid chemiluminescence enzyme immunoassay (CLEIA) was developed for the determination of microcystin-LR (MC-LR) by using strategies for oriented immobilization of functionally intact polyclonal antibodies on chitosan surface. Several physicochemical parameters such as metal ion adsorption, hexahistidine-tagged Protein G sorption, the dilution ratio polyclonal antibody concentration, and peroxidase-labeled MC-LR concentration were studied and optimized. The sorption in batch system of G-histidine and G-proteins was studied on a novel sorbent consisting of chitosan/divalent metal ions. Transition metals as Ni++and Zn++were immobilized through interaction with -NH2groups of chitosan in order to supply a material capable to efficiently remove the proteins from aqueous solutions. The maximum uptake of divalent metals onto the chitosan material was found to be 230 mg g-1for Zn++and 62 mg g-1for Ni++. Experimental data were evaluated using the Langmuir and Freundlich models; the results were well fitted with the Langmuir model; chitosan/Ni++foam was found to be the best sorbent for G-protein, maximum sorption capacity obtained was 17 mg g-1, and chitosan/Zn++was found to be the best for G-histidine with a maximum sorption capacity of 44 mg g-1. Kinetic data was evaluated with pseudo-first- and pseudo-second-order models; the sorption kinetics were in all cases better represented by a pseudo-second-order model. Under optimum conditions, the calibration curve obtained for MC-LR gave detection limits of 0.5 ± 0.06 μg L-1, the 50 % inhibition concentration (IC50) was 2.75 ± 0.03 μg L-1, and the quantitative detection range was 0.5-25 μg L-1. The limit of detection (LOD) attained from the calibration curves and the results obtained demonstrate the potential use of CLEIA with chitosan support as a screening tool for the analysis of pollutants in environmental samples.
authors.affiliation: École des Mines d'Alès, Centre des Matériaux des Mines d'Alès, 6 Avenue de Clavières, 30319, Alès CEDEX, France.
authors.forename: Hary
authors.initials: H
authors.lastname: Demey
authors.affiliation: École des Mines d'Alès, Laboratoire de Génie de L'Environnement Industriel, 6 Avenue de Clavières, 30319, Alès CEDEX, France.
authors.forename: Scherrine A
authors.initials: SA
authors.lastname: Tria
authors.affiliation: École des Mines d'Alès, Laboratoire de Génie de L'Environnement Industriel, 6 Avenue de Clavières, 30319, Alès CEDEX, France.
authors.forename: Romain
authors.initials: R
authors.lastname: Soleri
authors.affiliation: Department of Biomedical Engineering, The Dwight Look College of Engineering, Texas A&M University, 5045 ETB, College Station, TX, 77843, USA.
authors.forename: Anthony
authors.initials: A
authors.lastname: Guiseppi-Elie
authors.affiliation: École des Mines d'Alès, Laboratoire de Génie de L'Environnement Industriel, 6 Avenue de Clavières, 30319, Alès CEDEX, France. Ingrid.Bazin@mines-ales.fr.
authors.forename: Ingrid
authors.initials: I
authors.lastname: Bazin
mesh_terms: D000327:Adsorption; D055910:Antibodies, Immobilized; D048271:Chitosan / Q000737:chemistry; D004784:Environmental Monitoring; D006863:Hydrogen-Ion Concentration; D007124:Immunoenzyme Techniques; D007477:Ions; D007700:Kinetics; D008163:Luminescent Measurements / Q000379:methods*; D008387:Marine Toxins; D052998:Microcystins / Q000737:chemistry*; D012680:Sensitivity and Specificity; D013997:Time Factors
chemical_list: D055910:Antibodies, Immobilized; D007477:Ions; D008387:Marine Toxins; D052998:Microcystins; D048271:Chitosan; C057862:cyanoginosin LR
keywords: Affinity tag; Antibody orientation; Chitosan; Microcystin-LR; Protein G