Introduction A Background

Agglutination of particles was one of the earliest techniques used to detect antigen-antibody reactions. After mixing sample and reagent (as a colloidal suspension), the signal that reveals the reaction is the production of aggregates visible to the naked eye. Agglutination occurs either because when the molecule in the liquid phase binds its counterpart on the solid phase, it produces the linkage of particles (so the molecule must be at least bivalent), or because it generates a destabilization of the colloidal suspension by modifying the particle electric double layer (i.e., once bonded to the surface, the molecule decreases the surface potential).

The earliest particles used included charcoal, red blood cells, and bentonite. The classic immunoassay for the diagnosis of syphilis by agglutination of cholesterol particles has been used for many years in spite of serious limitations concerning its specificity. The continued preference for agglutination tests is due to certain advantages: they are easy and quick to perform, and do not require skilled personnel or sophisticated instruments. The first assays to be developed were "card tests" or "tube tests"; the reaction was performed on a card or in a test tube and the agglutination rated with the naked eye. With the introduction of radio- and enzyme immunoassays, which provide greater sensitivity, other techniques to evaluate the agglutination were developed. Nephelometric or tur-bidimetric measurement of agglutination not only improved sensitivity but also allowed the assays to be automated.

Since van den Hul and Vanderhoff's pioneer studies [1,2] on surface and size characterization of synthetic polymer colloidal particles, and their subsequent application to the detection of rheumatoid factors by Singer and Plotz [3], the use of such particles was quickly extended to a great many immunoassays [4,5]. Replacing "natural" particles with polymeric ones provides great advantages, especially the possibility of tailoring particles according to the exigencies of the assay under development. Moreover, synthetic particles are immunologically inert, whereas natural particles frequently give rise to nonspecific interactions. Latex particles were widely used in basic colloidal physics research as model colloids [6-10], and the development of this discipline has led to a better understanding of the physical chemical behavior of the latex-protein conjugate, especially where colloidal stability and the process of agglutination reactions are concerned [11]. A great many different polymers have been studied (polyvinyl-benzene, polymethacrylates, polystyrene, etc.), but polystyrene has been most extensively used for physical immobilization.

B. Immobilization of Immunoreagents

Classic physical adsorption of macromolecules as an immobilization procedure has some important advantages: it is easy to carry out, very reproducible, and suited to the production of large quantities of reagent. Immobilization occurs by the hydrophobic effect upon simple mixing of the latex with a solution of the macromolecule, under the right conditions of pH and ionic strength. This procedure has the additional advantage that a single type of solid phase particle can be used to immobilize a wide range of macromolecules, especially proteins. The most popular polymeric latexes, such as polystyrene, have highly hydrophobic surfaces and charged groups (-OSO3-) which stabilize the colloidal suspension. These groups are derived from the reagents used during synthesis (emulsion polymerization, where persulfate ions are used as a source of free radicals to initiate polymerization). Carboxyl groups may also appear by degradation of the sulfate ions (Kolthoff's reaction) [12]. In summary, a typical polystyrene latex possesses a highly hydrophobic surface with a low charge density, of the order of 3-5 pC/cm2. This charge has little or no influence on the establishment of final immobilization interactions; occasionally, when a mixture of proteins is being adsorbed, the surface charges can favor diffusion to the surface of a particular molecule [13], so that the final composition on the latex surface may be different from the initial composition of the protein mixture, especially if the amount of protein is well above what is required to saturate the latex surface.

However, physical adsorption does have some disadvantages—which rule out the development of some immunoassays—such as the following: (1) Some molecules are difficult to immobilize on hydrophobic surfaces because they are themselves highly hydrophilic, as in the case of polysaccharides and highly glycosylated proteins. Here one must resort to latex with highly charged and hydrophilic surfaces, which allow a large number of electrostatic interactions to occur. These interactions are fundamentally dependent on the pH and ionic strength of the medium, and if these parameters vary desorption of adsorbed molecules may occur, particularly if the conditions for adequate stability of the binding of the adsorbed molecule do not coincide with optimal pH and ionic strength for the antigen-antibody reaction. (2) The orientation acquired by molecules upon immobilization may prevent their recognition. Such orientation is linked to the distribution of hydrophobic patches (which provide the molecule's anchorage to the surface), with respect to its dominant epitopes (Fig. 1). It is difficult to manipulate the orientation of attachment solely by varying the physicochemical properties of the medium during immobilization. (3) Finally, physical adsorption is almost certain to cause denaturing of the macromolecules, since the adsorption process can alter the equilibrium of the forces that maintain secondary and tertiary structure [14-17]. The extent of denaturing may be quite variable, and it depends on the structural rigidity of the molecule [18] (Fig. 2) and the solid phase area available per molecule to be adsorbed (Fig. 3). Rigidity of the molecule depends essentially on the number of intramolecular disulfide bridges; non-covalent interactions also support the native structure, but they are more easily disrupted by the immobilization process. The larger the solid phase area available per molecule to be adsorbed, the greater will be the unfolding of the macromolecule, making more binding contacts with the surface, and the greater the extent of denaturation.

FIG. 1 Physical adsorption involves hydrophobic interactions between the surface and hydrophobic patches in the protein. It is a random process wherein the protein orientation is not the same for all of the immobilized molecules. As schematized in the figure, the way the protein is immobilized affects antibody recognition. Thus, in (a) antigen-antibody reaction is allowed, whereas in (b) the epitope is not exposed to the solution and the antigen-antibody reaction is avoided.

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