Immobilization of antibodies on alginate-chitosan beads (original) (raw)
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Journal of chromatographic science, 2018
High-affinity ligands, such as protein A, can be used to develop biocompatible matrices for antibody purification. In this paper, two methods were used for immobilization of protein A on the chitosan. In the first approach, amino groups of chitosan beads were functionalized with tris(2-aminoethyl)amine to produce amine double-branched moieties, which were subsequently activated with glutaraldehyde. In the second approach, chitosan beads were directly modified by glutaraldehyde to produce aldehyde groups. Structural characterization and successful modification of the functional groups on the supports were confirmed by scanning electron microscopy, FTIR spectroscopy and elemental analysis. Covalent immobilization of protein A was then performed on the surface of both supports. The immobilization yield was determined by using fluorescence spectroscopy, showing almost 15% increased capacity for the double-branched derivatized chitosan. The Immunoglobulin G (IgG) purification ability of ...
Covalent immobilization of antibodies for the preparation of immunoaffinity chromatographic supports
Separation Science and Technology, 2016
Immunosorbents in immunoaffinity chromatography (IAC) are prepared by immobilizing expensive antibodies without guidelines for ensuring the best coupling efficiencies, and avoiding low binding capacities. Covalent immobilization of antibodies on N-hydroxysuccinimide (NHS)-activated Sepharose 4 Fast Flow resin was optimized using human IgG via full factorial design with incubation times (4, 9, 14, 19 and 24 h), temperatures (4°C and 20°C) and coupling reaction buffers (sodium bicarbonate and triethanolamine). The best coupling efficiency (CE) (83.4 ± 8.7%) was reached with triethanolamine buffer, 14 h and 4°C. Comparison of antibody isotypes (IgG or IgM) by a nested factorial analysis suggested that antibodies in the IgG isotype presents the best coupling efficiency.
A novel support for antibody purification: Fatty acid attached chitosan beads
Colloids and Surfaces B: …, 2009
Linoleic acid attached chitosan beads [poly(LA-Ch)] (1.25 m in diameter) are obtained by the formation of amide linkages between linoleic acid and chitosan. Poly(LA-Ch) beads are characterized by FTIR, TEM, and swelling studies. Poly(LA-Ch) beads are used for the purification of immunoglobulin-G (IgG) from human plasma in a batch system. The maximum IgG adsorption is observed at pH 7.0 for HEPES buffer. IgG adsorption onto the plain chitosan beads is found to be negligible. Adsorption values up to 136.7 mg/g from aqueous solutions are obtained by poly(LA-Ch) beads. IgG adsorption saw an increase as a result of increasing temperature. Higher amounts of IgG are adsorbed from human plasma (up to 390 mg/g) with a purity of 92%. The adsorption phenomena appeared to follow a typical Langmuir isotherm. It is observed that IgG could be repeatedly adsorbed and desorbed without significant loss when we take into account the adsorption amount. It is concluded that the poly(LA-Ch) beads allowed one-step purification of IgG from human plasma.
Biomacromolecules, 2006
Antigens immobilized on solid supports may be used to detect or purify their corresponding antibodies (Ab) from serum. Direct immobilization of antigens on support surfaces (through short spacer arms) may promote interesting stabilizing effects on the immobilized antigen. However, the proximity of the support may prevent the interaction of some fractions of polyclonal Ab with some regions of the antigen (those placed in close contact with the support surface). Horseradish peroxidase (HRP) was immobilized on agarose by different protocols of multipoint covalent immobilization involving different regions of the antigen surface. Glyoxyl-agarose, BrCN-agarose, and glutaraldehyde-agarose were used as activated supports. Each HRP-immobilized preparation was much more stable than the soluble enzyme, but it was only able to adsorb up to 60-70% of a mixture of polyclonal anti-HRP antibodies. On the other hand, HRP was also immobilized on agarose through a very long, flexible, and hydrophilic...
The Chemical Engineering Journal, 1986
Attempts to scale up immunoaffinity separations have highlighted the limitations of the commonly employed CNBr-actiuated agarose gel supports. As an aid to the selection of an alternative configuration, we investigated the behaviour of a monoclonal antibody covalently immobilized to styrene, nylon and acrylic acid using several different chemical methods. The level of uptake of antibody was found to be dependent on the immobilization method used. The apparent antibody-antigen dissociation constant of the immobilized antibody averaged 1.5 X 1 OM6 M. This value was an order of magnitude lower than previously reported results for the same antibody in free solution and was not affected by either the immobilization method or the support polymer used. The different covalent bonds were subjected to commonly encountered eluting agents. Most were stable in at least one solution capable of breaking antibody-antigen bonds although all were highly labile in 4 M KI. The most stable bond in elutant was formed by glutaraldehyde-activated amino groups, followed by bonds formed through N-hydroxysuccinimide esters. Overall, the results suggest that immobilization through glutaraldehydeactivated amino groups, a simple procedure, results in a highly stable bond without adversely affecting antibody immunological reactivity.
Peptide immobilization on calcium alginate beads: applications to antibody purification and assay
Journal of Chromatography B: Biomedical Sciences and Applications, 1995
Two different procedures were developed for the non-covalent immobilization of peptide antigens on calcium alginate beads. The antigenic peptide is first synthesized in a multimeric form starting from a polydentate lysine core, and then immobilized on alginate beads (average volume 0.05 ml) by entrapment or simply by non-covalent adsorption. Coupling yields, as monitored by RP-HPLC analysis of the immobilization time course and/or by amino acid analysis of derivatized beads, were close to 1-2 mg of peptide per ml of gel. Immobilization yields were not dependent on the peptide net charge, hydrophobicity or length, but mainly on the extent of peptide multimerization. After immobilization on alginate gel, peptide antigenic properties were fully retained, as clearly demonstrated by the batchwise micropreparative purification of anti-peptide antibodies in good yields and with a high degree of purity, directly from crude sera in a single adsorption-elution step. Derivatized beads were sufficiently stable towards repeated washing-equilibration procedures, allowing very limited peptide leakage from the matrix. Peptide beads were also successfully used for the development of solid-phase immunoassays in test-tubes to characterize the corresponding antibodies, with the immobilization yield and signal-to-noise ratio being greatly enhanced in comparison with other types of conventional supports. * Corresponding author. 0378-4347/95/$09.50 © 1995 Elsevier Science B.V. All rights SSD1 0378-4347(94)00353-X
Methods, 2012
A method is described based on high-performance immunoaffinity chromatography for examining the interactions of immobilized antibodies or related binding agents with their targets. It is shown how this method can be used to obtain information on the binding, elution and regeneration kinetics of immobilized binding agents, such as those used with immunoaffinity supports. The theory behind this approach is briefly described and it is demonstrated how both the kinetic and thermodynamic properties of a biointeraction can be determined experimentally through this method. Several applications are used to illustrate this technique, including antibody-antigen interactions and the binding of aptamers with their targets in the presence of silica-based supports. The same approach can be adapted for use with other types of targets, binding agents and support materials.
2009
Pendent nitrile groups of multifilamentous polyacrylonitrile (PAN) fibers were reduced to amino groups using lithium aluminum hydride for different time of reduction and amine content was estimated by performing acid-base titrations. Attenuated total reflection-fourier transform infrared spectroscopy (ATR-FTIR) and Differential Scanning Calorimetry (DSC) were used for the characterization of the generated amino groups and thermal properties of the reduced fibers, respectively. The surface morphology of the fibers after reduction and immobilization was characterized using Scanning Electron Microscope (SEM). The newly formed amino groups of the fibers were activated by using glutaraldehyde for the covalent linking of Goat anti-Rabbit IgG-HRP (GAR-HRP) antibody enzyme conjugate. Modified PAN fibers were evaluated as a matrix for sandwich ELISA by using Goat anti-Rabbit antibody (GAR-IgG), Rabbit anti-Goat (RAG-IgG) as analyte and enzyme conjugate GAR-HRP. The fibers reduced for 24 h were able to detect the analyte RAG-IgG at a concentration as low as 3.75 ng mL −1 with 12% skimmed milk as blocking reagent for the optimized concentration of primary antibody GAR-IgG 3 g mL −1 and peroxidase conjugate GAR-HRP dilution of 8000 fold. The sensitivity, specificity and reproducibility of the developed immunoassay was further established with antibodies present in human blood using Rabbit anti-Human (RAH-IgG) antibody and the corresponding HRP enzyme conjugate. As low as 0.1 L of human blood was sufficient to perform the assay with the modified fibers.
Purification of antibodies with immunoadsorbents prepared using bromoacetyl cellulose
Immunochemistry, 1967
Al~tractmConjugates prepared by reacting bromoacetyl cellulose with proteins, synthetic polypeptides, and hapten derivatives of proteins and synthetic polypeptides have been studied for their immunoadsorbent properties. Each antigen showed differences in the extent of attachment to the cellulose and the ability of the antigen-cellulose conjugate to adsorb antibody. These conjugates had high capacity for extracting antibodies of the IgM and IgG classes of immunoglobulins from antisera. There was almost no non-specific interaction with other serum proteins at conditions at which the antibodies were bound to the immunoadsorbent. The immunoadsorbents were stable for several months and could be reused repeatedly without significant loss of their antibody binding capacity. The antigencellulose conjugates described may be useful as analytical reagents for the detection of antibodies as well as for isolation of specific immunoglobulins.
Immobilization of polyacrylamide-based glycoconjugates on solid phase in immunosorbent assays
Methods in molecular biology (Clifton, N.J.), 2012
Our experience in coating of solid surfaces with glycans, mainly for obtaining routine glycoarrays based on immunological plates, is summarized. Three polystyrene coating techniques are described: direct physical adsorption, covalent binding, and immobilization using the biotin tag. Protocols for studies on anticarbohydrate antibodies are considered, with special emphasis on the application niches of different immobilization techniques as related to the specificity of each method of glycan-binding protein assay, as well as the problems of background binding and quantitative estimation of the results.