Characterization of Immunoglobulin G Bound to Latex Particles Using Surface Plasmon Resonance and Electrophoretic Mobility (original) (raw)
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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.
Comparative Studies on Physically Adsorbed and Chemically Bound IgG to Carboxylated Latexes, II
Journal of Colloid and Interface Science, 1995
particles and simple coupling chemistries. There have been The aim of the present work is to compare the behavior of two general approaches to reagent stability: first, the binding physically (passively) adsorbed and of chemically (covalently) of antibodies to latex particles by noncovalent means is of bound IgG to carboxylated latexes (CLs). In this work we have such high affinity that little will ever leach off; second, on tried to determine the advantages and disadvantages of covalent a theoretical basis covalent coupling is worth pursuing as it coupling of antibodies to CLs against the passive adsorption proceshould provide a more stable reagent. There is little comparadure. Physically adsorbed IgG was measured by direct UV spectrotive data to provide a definitive answer to this problem. We photometry at 280 nm. Even when the protein has the same charge are unaware of any comparative study on the behavior of sign as its sorbent, adsorption occurs spontaneously, and this sug-IgG physically (passively) adsorbed and IgG chemically gests an adsorption caused by hydrophobic forces. IgG chemically bound using the carbodiimide method was both directly measured (covalently) bound to CL microspheres that indicates how using the bicinchoninic acid reaction (BCA method) and indirectly much specific activity of the IgG is preserved when it is calculated using UV spectrophotometry. The covalent coupling attached to CLs. This work is an attempt to determine the had an efficiency rate of more than 70%. The different electrophoadvantages and disadvantages of the covalent coupling of retic behavior shown by the IgG covalently bound to the CL parti-IgG molecules to CLs versus the passive adsorption procecles versus that of the IgG passively adsorbed on these particles dure, since the former has possible advantages, such as no is due to the presence of the carbodiimide used in those that are decrease in immunoactivity because of minimal denaturation chemically bound. Mobility values were dealt with using the most on the surface and because there is no desorption of antibodrecent theoretical approaches, given by Ohshima and Kondo, to ies, and minimal nonspecific interactions and a uniform couobtain information about the protein layer. The chemical coupling pling procedure (2). provides a better immunological response of the IgG-CL complexes.
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.
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
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.
Colloid & Polymer Science, 1992
An experimental investigation on the adsorption of immuno y-globulin molecules on polystyrene microspheres is described. Three different IgG samples were adsorbed on latex particles. One was of polyclonal nature with a broad range of isoelectric points (6.1-8.7), whereas the other samples were of monoclonal nature, Mab 1 and Mab 2 with i.e.p, of (5.65 + 0.15) and (7.7 + 0.I), respectively. Adsorption isotherms at different ionic strengths and pH were performed. Most of the adsorption isotherms showed well-defined plateaus. Because of instability in solution of Mab 2 in the pH values of 7 and 8, no plateau values were found in the adsorption isotherms at both pH-values. Maximum protein adsorption was found around the i.e.p, of the protein. According to the findings, the IgG adsorption on polystyrene surface is strongly irreversible with respect to pH changes. The ionic-strength changes, however, exert a pronounced effect on the adsorption-desorption processes of IgG on negatively charged polystyrene surface. Also, electrophoresis experiments were performed to gain information on the electrostatic interaction between the IgG molecules and the PS latex. With increasing the adsorbed amount of IgG the absolute value of mobility decreases to reach a plateau value. The isoelectric pH of the IgG-PS complex is always smaller than the i.e.p, of the dissolved IgG, indicating that the PS surface charge must partly compensate the positive charge on the protein. Finally, the colloidal stability of the rabbit IgG/PS complex is always very low, whereas the Mab/PS complexes are very stable when the charge electrokinetically mobilized by these systems is very large.
Adsorption of Immunoglobulin G on Core-Shell Latex Particles Precoated with Chaps
Journal of Colloid and Interface Science, 2000
The aim of this work is to investigate the adsorption behavior of a monoclonal antibody (immunoglobulin G, IgG) on latex particles, possessing reactive chloromethyl groups, precoated with 3-([3cholamidopropyl]dimethylammonio-1-propanesulfonate (Chaps). The amount and reactivity of the surface chloromethyl groups were monitored by the nucleophilic attack of glycinate to the functional groups as a function of time at 22 and 36 • C. The extent of displacement of Chaps by IgG and the enthalpy of the process were determined under two different conditions of precoating the latex particles with Chaps, at 22 and 36 • C. The adsorption of IgG takes place in two steps; the first one involves physical interaction between IgG and the surface. This step is relatively fast (in the range of minutes) and independent of temperature. In the second step covalent bonding between the protein and the active surface groups occurs. This reaction is improved by raising the temperature because Chaps desorption, which exposes the reactive chloromethyl groups on the latex particles, is kinetically and thermodynamically favored at 36 • C and the covalent bonding of IgG is faster at 36 • C.
Development of a high sensitivity IgG-latex immunodetection system stabilized by hydration forces
Polymer International, 1999
We present the application of hydration forces to obtain high sensitivity IgG±latex immunosystems. To compare these with another standard system, a study is presented of IgG± and F(ab') 2 ± latex conjugates in terms of colloidal stability and immunoreactivity. The stability domains have been examined using a low-angle light scattering technique (nephelometer). The protein-coated particles present an anomalous stability at high ionic strength when the classical theory predicts aggregation, and this stabilization is attributed to hydration forces. Different electrolyte concentrations and counterion valences have been tested to determine the most in¯uential factors on this stabilization mechanism. Long-term aggregation of the conjugates has also been studied by measuring the aggregate size by photon correlation spectroscopy. To quantify the immunoresponse of the agglutination tests, aggregation in the presence of antigen is followed as a function of time with the nephelometer. The considerable increase in immunoresponse, together with the decrease in possible perturbing side-reactions enhances the technical interest of this method of stabilizing immunolatexes.
A comparative study between the adsorption of IgY and IgG on latex particles
Journal of Biomaterials Science-polymer Edition, 2000
The use of egg yolk antibodies(IgY) instead of IgG from mammalian species may present several advantages in the development of routine diagnostic immunoassays. On the one hand, the animal suffering is reduced, as antibodies are obtained directly from the egg. On the other hand, the use of IgY avoids the rheumatoid factor interference. The rheumatoid factor interacts with IgG molecules in many immunoassays causing false positive results. Despite these advantages, IgY antibodies are scarcely used. As part of an aim to develop a diagnostic test based on IgY-latex agglutination, a preliminary study on some characteristics of the IgY-latex complexes is carried out. In this work, protein adsorption and desorption, isoelectric point, electrokinetic mobility, and colloidal stability are analysed. Results are compared to those obtained by IgG. Interesting differences are observed (which mainly arise from the difference in molecular structure between IgY and IgG), suggesting that IgY is a more hydrophobic molecule than IgG. In addition, colloidal dispersions of IgY-covered latex particles are more stable (at pH 8) than those sensitized by IgG.