Sorption of his-tagged Protein G and Protein G onto chitosan/divalent metal ion sorbent used for detection of microcystin-LR

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 –NH₂ groups 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⁻¹ for Zn⁺⁺ and 62 mg g⁻¹ for 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.