Journal of Colloid and Interface Science 245, 230–236 (2002) doi:10.1006/jcis.2001.7958, available online at http://www.idealibrary.com on Interaction of Bacterial Endotoxine (Lipopolysaccharide) with Latex Particles: Application to Latex Agglutination Immunoassays J. M. Peula-Garc´ ıa, ∗ J. A. Molina-Bolivar, ∗ J. Velasco,† A. Rojas,‡ and F. Galisteo-Gonz´ alez§ ,1 ∗ Departamento de F´ ısica Aplicada II, Universidad de M ´ alaga, 29071 M ´ alaga, Spain;†Laboratorios Ovejero S.A., 24008 Le´ on, Spain;‡Vircell S.L., Pol. Industrial “Dos de Octubre,” 18230 Santa F´ e, Granada, Spain; and §Departamento de F´ ısica Aplicada, Universidad de Granada, 18071 Granada, Spain Received January 8, 2001; accepted September 17, 2001 The latex agglutination immunoassay technique uses polymer colloids as carriers for antibodies or antigens to enhance the im- munological reaction. In this work, the interaction of a lipopolysac- charide (LPS) of Brucella Melitensis with two conventional latexes has been studied. Some experiments on the physical adsorption of the LPS onto these polystyrene beads have been performed and several complexes with different coverage degrees were obtained by modifying the incubation conditions. Regarding the application in the development of diagnostic test systems, it is advisable to study the latex–LPS complexes from an electrokinetic and colloidal sta- bility point of view. The complexes were electrokinetically charac- terized by measuring the electrophoretic mobility under different redispersion conditions. The colloidal stability was determined by simple turbidity measurements. Experimental and theoretical data have been employed to study the molecular disposition of the LPS in the latex particle surface to compare with the outer membrane of bacterial cells. Latex complexes covered by different LPS amounts showed high colloidal stability and adequate immunoreactivity that remains for a long time period. C  2002 Elsevier Science Key Words: polystyrene latex; LPS; electrokinetic characteriza- tion; particle-enhanced immunoassays. I. INTRODUCTION The interaction between biomolecules and latexes has con- siderable importance in the development of particle-enhanced immunoassays. The use of synthetic polymer colloids for sero- logical diagnosis is normally possible by coupling of ligands, antigens, or antibodies to the surface of particle latex. Several procedures for physical and covalent coupling of proteic ligands have been previously described (1–5). However, the antigenic characteristics for a great number of microorganisms do not re- side in protein molecules, making very interesting the study of the interaction between non-proteic ligands and latex particles. Lipopolysaccharide (LPS), a major constituent of the outer membrane of gram-negative bacteria, is the main antigen of these bacterial cells (6). Interaction of LPS with latex particles is of great interest since it allows the development of rapid serological 1 To whom correspondence should be addressed. E-mail: galisteo@ugr.es. immunodiagnostic tests (7, 8). In these works, however, colloidal properties of the latex complexes obtained are not studied, when it is very important for their practical application. The adsorption of a ligand is a complex process in which several physico-chemical factors are involved. The amount and way in which the ligand is adsorbed depends on the nature of the ligand, the characteristics of the solid surface, and the solution conditions. The LPS molecule, with two well-defined parts (hydrophobic and hydrophilic), may interact with latex particles to render surface complexes similar to those of the bacterial outer membranes. In this work, we have used two latexes with different particle sizes to study the coupling of Brucella Melitensis LPS. Adsorp- tion experiments with different solution conditions of pH and ionic strength have been performed to obtain different LPS–latex complexes. These biointerfaces were characterized from an elec- trokinetic point of view to study the change in the electrical state of the particle covered by LPS as well as to obtain information about the possible configuration of this molecule on the surface. Another important characteristic of these complexes is their colloidal stability that prevents particle aggregation in the absence of specific antibodies. We have studied the conditions in which these complexes are colloidally stable. This property will be useful to check if these LPS–latex systems can be used for immunological applications. In this way, the presence of specific Brucella antibodies in rabbit and human serums was proved by the immunoagglutination of the complex particles in different serum dilutions. II. MATERIALS AND METHODS All chemicals used in this study were of analytical grade and were used without further purification. Water used in all experi- ments was doublly distilled and deionized with a Milli-Q water purification system (Milipore). Two polystyrene latexes were employed in this study: a sul- fonate latex obtained by a “shot-growth” emulsion polymeri- zation technique, using sodium styrene sulfonate as the como- nomer (latex A) (9), with an average particle diameter of 195 ± 10 nm and a surface charge density of −4.2 ± 0.6 μC/cm 2 , and a sulfate sample obtained by a surfactant-free emulsion 230 0021-9797/02 $35.00 C  2002 Elsevier Science All rights reserved.