Many kinds of functionalized latexes with reactive surface groups suitable for covalent protein immobilization have been described. Some of the more common choices include the following:
The most commonly used surfaces for attachment of ligands are carboxyl and amino groups, for the following reasons:
1. These groups have proven to be very stable over time.
2. The chemistries involved in attaching ligands to either of these groups have been widely explored, and several options exist for each.
3. The existence of terminal amino and carboxyl groups on proteins is universal, ensuring their availability for complementary attachment to one or the other functional group on the surface of the microspheres.
A number of special linkers can be used to convert one surface functional group on a microsphere to another. For example, amino microspheres can be converted to carboxylic particles by reacting with succinic anhydride . Conversely, carboxylic particles can be converted to amino microspheres through water-soluble carbodiimide-mediated attachment of a diamine . Also, sulf-hydryl particles can be made by reacting amino particles with iminothiolane .
The functionalized groups may be used as sites for the attachment of spacer arm molecules. These spacer arms have functional groups at the distal end for the flexible bonding of proteins. In this way, antibody molecules extend away from the latex surface into the aqueous medium. This approach may minimize
protein denaturation and the antigen-antibody recognition could be easier because the coupled antibodies are set off from the surface [64,65]. For example, it is possible to attach amine spacer arm molecules to activated chlorine groups at the latex surface.
Some functional groups borne by the latex are unreactive as such and need to be activated prior to protein immobilization: cyanogen bromide is used to bind hydroxyl groups from the latex surface to amine groups in the protein at alkaline pH, and glutaraldehyde is used to link amino groups present on the latex to amino groups on the antibody molecule. Most of the work on the cova-lent binding of proteins has been conducted on carboxylated latexes [66,67]. The methodology required for coupling proteins to unactivated latexes is tedious (involving several steps before the activated groups react with free groups of the protein), expensive, and more time consuming than one-step coupling. The carboxyl groups have to be activated by the 1-ethyl-3-(3-dimethylaminopro-pyl)carbodiimide (EDAC). The intermediate obtained on reaction of a carbox-ylic acid group with a carbodiimide is fairly unstable, especially in water, and has to be quickly mixed with the protein to be immobilized. This is the major drawback of any immobilization protocol based on the use of highly reactive and unstable intermediates. The balance between covalent coupling and unproductive side reactions depends to some extent on activation reaction conditions. The ionic composition of the reaction, the pH, and the buffer type can greatly enhance or inhibit the total binding of protein to particles. A number of variations exist for EDAC-mediated coupling. The degree of coupling is dependent on the density of the reactive groups, i.e., carboxyl groups on the polymer. Sufficient amounts of functional groups should be present to provide adequate coupling of antibody. The covalent attachment of the IgG molecule to carboxyl-ated particles improves the immunoreactivity of antibodies when compared with physical adsorption, maintaining its immunoreactivity after long periods of storage .
Some authors have described the preparation of diazotized polystyrene for use in the separation and purification of antibodies [69,70] and for latex agglutination testing . This functionalized latex with active diazo groups can be coupled to phenol and imidazole groups of antibodies via a covalent bond linkage. This immunopolystyrene diazonium latex reagent showed a positive agglutination reaction of 78-91% when mixed with serum from patients with lep-tospirosis.
Different authors have indicated that the use of aldehyde groups could simplify the covalent bond of the protein due to the direct reaction between the aldehyde groups of the latex and the primary amino groups in the protein molecules [72,73] by forming an imine derivate with concomitant water elimination. Rembaum et al.  described polymerization of acrolein to prepare micro-spheres that could be used as substrates for immobilization of proteins, but the microspheres were porous. Bale et al.  proposed a method for providing beads nonporous since reaction between an immobilized ligand and other reac-tant is expected to be faster on nonporous surfaces due to diffusional considerations. Alternatively, preformed polymeric latex could be modified to contain aldehyde groups .
In general, hydrophilic surfaces may have a lower level of nonspecific interactions than hydrophobic surfaces [77,78]. In this sense, Koning et al. 
proposed the synthesis of core-shell particles containing a hydrophilic polymeric shell with aldehyde groups. These particles have been used to detect human chorionic gonadotropin (HCG) in urine and serum. The results were compared with hydrophobic latex particles with the same antibodies physically adsorbed. The results showed that the functionalized latex presents a less nonspecific interaction and a higher detection limit. Hydrophilic particles with functional aldehyde groups can be produced by polymerization of glutaraldehyde and of acro-lein at high pH . New approaches can be accomplished by producing particles with a uniform distribution of functional group areas separated by hy-drophilic areas [81,82]. The former can be used for attaching proteins, the latter for inhibiting nonspecific effects. Ideally, particles should be available with different percentages of the two types of areas in order to optimize assay concentration ranges.
The aldehyde groups tend to decompose with time, losing the capacity to bind the proteins. As suggested by Kapmeyer et al. [83,84], a possibility is to produce latex particles with acetal groups on the surface. These groups can be transformed to aldehyde groups at the moment to produce the covalent coupling of the proteins, by moving the medium to acid pH. Peula et al. [85-87] prepared acetal latexes, which permitted the covalent coupling of IgG anti-C-reactive protein (anti-CRP) in a simple way by changing the pH of the suspension to pH 2. The latex-protein complexes showed a good immunological response that was not disturbed by the presence of a nonionic surfactant in the reaction medium and was stable with time.
Preactivated microparticles have been developed with surface groups, which are sufficiently reactive to directly couple with proteins. There is no requirement for a separate preliminary activating step. This convenience allows fewer handling and transfer steps. An example of preactivated groups is vinylbenzyl chloride [88-90], where the reaction occurs by nucleophilic displacement of the chloride atom of chloromethylstyrene groups. Such latexes have limited shelf life due to the inevitable hydrolysis or oxidation of the reactive groups in aqueous media. The stability of chloromethyl function is strongly dependent on temperature. The hydrolysis rate increases with increasing temperature. At the storage temperature of 4°C some hydrolysis occurs, and after a long period of time (1 year) approximately 20% of the surface chloromethyl groups disappear . Nevertheless a significant proportion of the reactive groups are retained. A chlo-romethylstyrene monomer can be polymerized onto a polystyrene core in any proportion to other nonactivated containing monomer, to produce a shell having from 5% to 100% chloromethylstyrene monomers .
Sarobe et al.  have studied the covalent immobilization of lysozyme on chloromethyl latexes. As can be seen in Fig. 10, the initial steps of the adsorption isotherm indicate that all the adsorbed protein becomes covalently bound. This result is general if a certain surface density of functional groups exists on
the surface (>21 pmol/g polymer). As physical contact occurs prior to chemical linking, a sufficiently high number of chloromethyl groups are needed to ensure that covalent binding can take place. With more adsorbed protein at the surface, the covalent extent decreases to a more or less constant value between 60% and 70% independent of the number of chloromethyl groups and pH.
A method to show the existence of covalency between protein and functional-ized latex is treatment of protein-latex system with surfactants under appropriate conditions able to recover all the physically adsorbed protein from the particle surface. In the case of chloromethyl latexes, the determination of free chloride ions after protein adsorption and comparison with a blank could show the covalent attachment (Fig. 11). The kinetic of the aminochloromethyl reaction at the interface is slow. Although physical adsorption achieves saturation after some minutes of sensitization , covalent binding is quite slower, needing several hours to be completed (5-6 h). This means that the first contact between the protein and surface is always physical, while chemical linking develops later. Nustad et al.  demonstrated that adsorptive binding to core-shell particles occurred rapidly followed by slow covalent coupling. It has been claimed that adsorption is a necessary prerequisite to covalent coupling and that
FIG. 11 Evolution of chloride ion release as a function of sensitization time for protein-saturated conditions.
FIG. 11 Evolution of chloride ion release as a function of sensitization time for protein-saturated conditions.
an excessively high charge density on the latex surface will actually decrease the yield of covalent coupling .
In practice, with preactivated microparticles a blocking step in the process of preparing antibody-coated latex has to be included to eliminate the unreacted functional groups (in the case of chloromethyl groups with inert amines). Figure 12 shows a functionalization scheme of chloro-activated latexes to obtain functional groups other than chloromethyl groups through a linked spacer arm and without a linked spacer arm .
The immunoreactivity of IgG or F(ab')2 antibody molecules covalently bound to the surface of chloro-activated latex has been compared to passive physical adsorption to a conventional polystyrene latex [98,99] (Fig. 13). For both antibodies an improvement in the immunoresponse is observed for the covalent union to latex particles. The desorption of physically attached protein from the surface with time reduces the period for which latex agglutination tests may be stored. Molina et al.  indicated that the storage period for IgG and F(ab')2 antibodies covalently attached to chloro-activated surface is higher than for physical adsorption.
In covalent coupling of proteins to functionalized latexes a variety of conditions need to be tested for each case. In many of the coupling procedures extensive multipoint binding takes place during covalent attachment of the proteins and latex particle because there is a very large number of functional groups on the particle surface. This multiple binding may render either the antigen or the antibody inactive. The binding procedures have to be adapted to minimize the protein denaturation.
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