Covalent coupling of antibodies to aldehyde groups on polymer carriers (original) (raw)
Related papers
Journal of Colloid and Interface Science, 1998
immunodiagnostic tests. The recent advances in polymer In this work the interaction of an a-CRP IgG protein with science have made it possible to produce uniform latexes functionalized latexes that have acetal groups on their surfaces with desired size and surface characteristics. Thus, it is possihas been studied. Two acetal latexes with similar amounts of ble to obtain latex particles with specific functional groups surface acetal groups but different surface charge densities were which can covalently bind antibody molecules via amine used. Some experiments on the physical and chemical adsorption groups. From the point of view of its immunological applicaof the IgG onto these polystyrene beads have been performed, tion, the covalent bond of proteins to the particle surface and several latex-protein complexes with the IgG physically or prevents the physical desorption of the proteins and retains chemically bound to the surface were obtained by modifying the the native conformation of these biomolecules. Most of the incubation conditions. In the covalent coupling experiments of the IgG, the physically adsorbed protein was removed by redisper-studies of covalent coupling of proteins were developed with sion of the complexes in the presence of a nonionic surfactant carboxylated latexes and require some chemical reaction (1-(Tween 20). After this treatment the final amount of protein on 3). Recently, several authors (4-7) indicated that the use of the latex surface was around 80% of the total protein initially aldehyde groups on the surface could simplify the covalent adsorbed. The latex-protein complexes that formed were characbinding of the protein due to the direct reaction between the terized from the electrokinetic point of view by measuring their aldehyde groups of the surface and the amine groups of the electrophoretic mobilities versus the pH, in order to detect any protein molecules. However, due to the chemical unstability difference between the particles when the protein is physically or of the aldehyde groups, they tend to decompose with time, chemically coupled. The isoelectric point ( iep ) of the complexes losing their capacity to link the proteins. As suggested by was around pH 4, where they will be unstable because the electro- Kapmeyer et al. (8,, another possibility is to produce latex static repulsion cannot stabilize the particles. At neutral and basic particles with acetal groups on the surface. These groups can pH, the electrophoretic mobility values of the latex-protein particles seem to predict a good colloidal stability which is a very be transformed to aldehyde groups to produce the covalent important aspect when looking for its application in the field of coupling of the proteins, by moving the medium to acid pH.
Chloroactivated latex particles for covalent coupling of antibodies. Application to immunoassays
Journal of Biomaterials Science, Polymer Edition, 1997
The aim of the present work is to prepare and characterize a functionalized latex with chloromethyl groups on the surface and to perform the covalent coupling of anti-human serum albumin (a-HSA) IgG protein. The chloromethyl-styrene latex (CMS) was synthesized by means of a core-shell emulsion polymerization in a batch reactor. The monodisperse-obtained latex was characterized by determining the diameter (TEM and PCS), the surface charge density (conductometric and potentiometric titration), the amount of chloromethyl groups on the surface (hydrolysis reaction), and the stability vs electrolyte concentration (turbidity measurements). Electrokinetic characterization was also performed (electrophoretic mobility versus pH and ionic strength). IgG was chemically bound to the latex particles under different sensitization and block-stabilization conditions. Colloidal stability of complexes was studied to select an immunolatex suitable for the development of latex immunoassays. The final part of this work consists of a study of the immunoreactivity of the IgG-latex complexes at different pH and ionic strength, in particular under physiological conditions. The results show that chemically bound IgG to chloromethyl latex provides an IgG-latex complex suitable for application in immunodiagnosis tests.
Journal of Colloid and Interface Science, 1998
The main objective of this work was the investigation of passive adsorption and covalent coupling of a polyclonal IgG and a monoclonal preparation of IgG against HSA, to a carboxyl latex particle. The functional activity of the coupled protein was then assessed by quantitative immunoassays for the antigen. Sensitized particles, with different protein coverage, were fully characterized using a range of different technologies, including electrophoretic mobility (e), photon correlation spectroscopy, and surface plasmon resonance (SPR). The antibody-labeled particles were studied with respect to electrokinetic behavior in pH and ionic strength titration, stability, antibody functionality, and their perfomance in immunoaggregation reactions. Important differences were observed between the two sets of particle preparations throughout the series of experiments. The differences could be attributed to the coupling of the IgG molecules to the particles by the two different adsorption protocols. When proteins were chemically bound to the polymer surface it was necessary to activate the carboxyl groups with a carbodiimide (CDI) moiety that in our case was positively charged. The differences in characteristics between the adsorbed and the coupled antibody particles are thought to be due to the fact that in the covalent coupling protocol some CDI molecules remained linked to the particles, which altered the average electrical state of the outer layer in comparison with those samples where antibodies were physically adsorbed. On the other hand, the isoelectric point of the monoclonal antibody was lower (5.4 ؎ 0.1) than the pI of the polyclonal antisera (6.9 ؎ 0.9), which could explain why the IgG-latex complexes created with monoclonal molecules were colloidally more stable at neutral pH than those created with the polyclonal antisera. However, no immunoaggregation of antibody particles by the presence of antigen was found with the former. The use of SPR demonstrated that the equilibrium constants for the antibody-antigen recognition of the two antibody preparations were quite similar (K A polyclonal IgG ؍ 2.8 10 8 M ؊1 ; K A monoclonal IgG ؍ 9.5 10 7 M ؊1). These observations suggest that the lack of aggregation mediated by antigen demonstrated by the monoclonal antibody coupled to the latex particles may be due to this protein recognizing only one epitope in the HSA molecule. However, as the repulsive charge between antibodylatex particles counteracts the attractive forces between the antigen epitope and the antibody paratope, the greatest immunoag-gregation was obtained when using latex particle-antibody complex with a low charge density (N) in the external layer.
Journal of Molecular Recognition, 1995
The authors have recently described the development of a carboxymethyl dextran-based sensor surface for biospecific interaction analysis by surface plasmon resonance. Ligands are immobilized via primary amine groups after activation of the carboxymethyl groups on the sensor surface with a mixture of N-hydroxysuccinimide and N-ethyl-N '-(dimethylaminopropyl) carbodiimide. Methods have now been developed for efficient immobilization via thiol/disulfide exchange, aldehyde coupling and biotin-avidin coupling. The specific activity of monoclonal antibodies immobilized by the four different methods was investigated by altering the immobilization conditions, e.g., activation time, protein concentration, ionic strength and the degree of modification, etc. Investigations have also been made concerning possible differences in the specific activity for antibodies immobilized using optimized conditions with respect to the four different chemistries. These studies show that, with the flexible carboxymethyl dextran matrix used here, the immobilization methods give rise to only minor differences in specific activity. Thus, with this solid support, a 'site directed' immobilization strategy for monoclonal antibodies has no advantage. In general the specific acitivity for optimized systems was approximately 75% for the binding of /32pglobulin to an immobilized monoclonal antibody directed against B;?p-globulin. Reduced specific activities of immobilized antibodies induced by variation of the coupling conditions could be attributed to the deterioraton of the active site of the antibody.
1997
immunodiagnostic tests. The recent advances in polymer In this work the interaction of an a-CRP IgG protein with science have made it possible to produce uniform latexes functionalized latexes that have acetal groups on their surfaces with desired size and surface characteristics. Thus, it is possihas been studied. Two acetal latexes with similar amounts of ble to obtain latex particles with specific functional groups surface acetal groups but different surface charge densities were used. Some experiments on the physical and chemical adsorption which can covalently bind antibody molecules via amine of the IgG onto these polystyrene beads have been performed, groups. From the point of view of its immunological applica-and several latex–protein complexes with the IgG physically or tion, the covalent bond of proteins to the particle surface chemically bound to the surface were obtained by modifying the prevents the physical desorption of the proteins and retains incubation conditions. ...
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.
Carboxylated Latexes for Covalent Coupling Antibodies, I
Journal of Colloid and Interface Science, 1995
polymerization are negatively charged in a broad pH range The aim of the present work is to prepare and characterize of 3-10 (4), but their surface charge density is extremely carboxylated latexes (CLs) in order to bind antibodies covalently high (4). At a low ionic strength the antibody cannot peneand efficiently. Carboxylated latexes were prepared by surfactanttrate the double layer of the particle and thus, no coupling free emulsion polymerization using an azo-initiator, which prooccurs (11). The carboxylated latexes prepared by an azovides carboxyl end groups on the latex surface directly. Two latexes initiator have a rigid interface and the surface charge density were characterized using different techniques: particle size by is significantly lower. These types of particles have been electron microscopy and photocorrelation spectroscopy, surface used less in covalently coupling antibodies and antigens to charge density by conductimetric and potentiometric titrations, polymer carriers in the development of particle-enhanced and electrophoretic mobility versus pH and ionic strength (KBr). The colloidal stability of both latexes shows that these carboxylated optical immunoassays. latexes can be used for biomedical applications. Activation of car-In this paper, we report the preparation of surfactant-free boxyl end groups was performed using the carbodiimide method, carboxylated polymer latexes which enables the covalent and the best conditions for covalent coupling of IgG were obtained. coupling of antibodies with a high efficiency rate. The elec
Biomacromolecules, 2008
The correct immobilization of antibodies is one of the most critical steps in the preparation of immunosensors and immunochromatography matrices. In addition, the final support has to be chemical and physically inert to avoid the unspecific adsorption of proteins that can reduce the sensitivity of the biosensor or the purification achieved by the chromatography. The solution to both problems is one of the major challenges in the field. Here, we have presented two different novel and simple alternatives to have the unmodified antibody anionically exchanged to a support, further covalently immobilized with more than 90% of the antibodies bonded to the support by the four subunits, retaining a high functionality and giving a final "inert" surface. The first solution was the use of supports having a low superficial density of amino groups activated with glutaraldehyde. Here, the inertness was achieved by the use of a very low density of amino groups, unable to adsorb proteins at 100 mM sodium phosphate, while immobilization proceeds mainly via a first adsorption of the antibody and a further reaction with the glutaraldehyde groups. The second solution implies the design of a novel support (aminoepoxy). This support again produces a first ionic exchange of the antibody on the support and a further reaction with the epoxy groups, but because the epoxy groups can be finally blocked with aspartic groups (annulling the charge), the initial density of amino-epoxy groups can be as high as possible. Both systems permitted the correct and oriented immobilization of IgG. The immobilized antibody showed high-functionality (65-75%) and a final inert support surface. This immobilized antibody (antiperoxidase) was able to capture fully specifically HRP contaminating a protein crude extract from E. coli.
Colloids and Surfaces A: Physicochemical and Engineering Aspects, 1994
In this work the competitive coadsorption of immunoglobulin IgG and bovine serum albumin (BSA) proteins on a sulfate polystyrene latex has been studied. The competitive coadsorption experiments were carried out by changing the initial concentration of both proteins (Cm-BSA] or [IgG/a-CRP]) in order to cover different possibilities of competition. By changing the pH and the ionic strength during the incubation, it was possible to obtain latex-protein complexes with different degrees of coverage by each protein. These complexes were characterized electrokinetically under several different redispersion conditions and their colloidal stability was also determined. By combining both results it is possible to establish that, at neutral pH, electrostatic repulsion is the main factor governing the colloidal stability. This stability appeared when negatively charged BSA molecules constituted the majority protein on the particle surface. However, the immunoreactivity of the sensitized latexes was sufficient with a partial coverage by IgG molecules. Therefore the properties of the latex-protein complex depended on the percentage of BSA or IgG adsorbed and on the charge state of the proteins at the redispersion pH. By using specific incubation conditions, latexes covered by different percentages of IgG/BSA were obtained, which showed high colloidal stability and good immunoreactivity.
Surface immobilisation of antibody on cyclic olefin copolymer for sandwich immunoassay
Biosensors and Bioelectronics, 2009
In this work, the surface functionalisation of the commercially available cyclic olefin copolymer (COC) materials, Zeonor R and Zeonex R , has been studied. The methodology employed involved oxidation in oxygen plasma, functionalisation of the oxidized surface with aminopropyl triethoxy silane and, finally, attachment of antibody using covalent linker molecules. 1,4-Phenylene diisothiocyanate was selected as the most suitable cross-linker for the attachment of protein, as assessed by fluorescent intensity measurements on immobilised FITC-labelled IgG antibody. The modification method was characterised by contact angle measurements, ellipsometry, X-ray photoelectron spectroscopy (XPS) and fluorescence microscopy. The data are consistent with the deposition of a polymeric film of the silane chemisorbed to the oxidised plastic surface. The functionalised surfaces were employed in a sandwich immunoassay format using the reagents goat anti-human IgG (G␣HIgG) and fluorescently labelled G␣HIgG (Cy5-G␣HIgG) as capture and detection antibodies, respectively, and with human IgG (HIgG) as the model analyte. The lowest concentration of HIgG detected was 0.1 ng ml −1 , with a relative standard deviation of 15%. Non-specific binding effects were also assessed. The method and supporting data demonstrate that simple approaches to surface functionalisation can be adapted to plastic-based devices.