Improvement Of Assays By The Development Of New Immunoreagents And Surfaces

In order to overcome the limitations described in the introduction, new immuno-assay techniques have been developed, with new methods for improving antigen reactivity and new surfaces to obviate the problems of physical adsorption.

A. Improvement of Specificity by Improving Immunoreagents

Classical biochemical methods for purifying antigens (such as a pathogen) from biological material give rise to samples that frequently contain other macromole-cules, which are of no value in the assay and which interfere with it considerably because they are not inert. They have a negative effect on the detection limit and the specificity of the test. A large part of the improvement in immunoassay performance has been due to improvements in the reagents as a result of attempts to immobilize only those molecules that are involved in the reaction of interest, thus avoiding the problems mentioned above. This has been achieved by using monoclonal antibodies, and/or molecularly defined antigens, or fragments of antigens. With the advent of genetic engineering [20], it became possible to synthesize perfectly defined proteins, which gave a highly specific signal in immunoassays. This versatile technology allows expression in bacteria of protein fragments of diagnostic value, which may contain linear or conforma-tional epitopes; sequence modifications can also be introduced to facilitate immobilization, whether by adsorption or by covalent linkage.

Recently, synthetic peptides have become another alternative in improving immunoassays [21]; these are linear amino acid polymers, made by chemical synthesis [22]. The study of these compounds did not arise from the need to improve immunoassays, but from basic research into the structure of epitopes [23]. Peptides of diagnostic value are B epitopes, i.e., they react with antibodies generated against proteins (immunogens) recognized as foreign to the organism (antigens) (Fig. 4).

From the point of view of assay production, incorporating synthetic peptides into immunoassays has definite advantages. The cost of reagents may be lower because tedious purification processes starting from complex biological materials are avoided and/or because the availability of very versatile automatic pep-tide synthesizers has allowed extraordinary decreases in the price of peptides in recent years. Production processes have also been simplified (economically as well as technically) and have benefited from the use of more stable, chemically pure, and reproducible compounds. Once production specifications have been defined, products can be manufactured almost indefinitely, in contrast with biological products, which tend to have batch-to-batch variations. Furthermore, synthetic peptides can be easily transported and stored, so that strict control of the properties and use of the final product is possible.

Peptides used in immunoassays typically contain from 7 to 25 amino acids; they may or may not belong to the primary sequence of the native protein, but even so they maintain their reactivity with specific antiprotein antibodies. Basically the length of a peptide should be that of a B epitope—in other words, the number of amino acids recognized in the interaction with an immunoglobulin

FIG. 4 Synthetic peptides as B epitopes. B epitopes, generally classified as linear (a, b) and conformational (c), are schematically shown in both the folded protein and the corresponding portions on its primary structure. Only linear epitopes are easily reproduced by synthetic peptides. This can be achieved either directly (a) or through modifications of the original amino acidic sequence in order to mimic the structure it adopts within the folded protein (b).

FIG. 4 Synthetic peptides as B epitopes. B epitopes, generally classified as linear (a, b) and conformational (c), are schematically shown in both the folded protein and the corresponding portions on its primary structure. Only linear epitopes are easily reproduced by synthetic peptides. This can be achieved either directly (a) or through modifications of the original amino acidic sequence in order to mimic the structure it adopts within the folded protein (b).

paratope. Crystallographic studies of the antigen-antibody complex have shown that the interactions involve about 15 amino acids [24,25]. In practice, a greater number of amino acids are frequently required; although they do not participate directly in the interaction, they contribute to overall structure and so favor recognition [26].

Two strategies have been developed for searching for peptides to substitute for the reactivity of a given antigen [27]: (1) primary sequence analysis of the target protein [28-30], and (2) random generation of amino acid sequences that are then selected according to their reactivity with antibodies raised against the antigen [31,32]. The first method starts from knowledge of the primary sequence of the protein of diagnostic value; epitopes are predicted by algorithms [33,34] or experimental methods [35]. The algorithms used to analyze the primary sequence select sequences that have high probability of being exposed on the surface of the protein and have certain exposure, flexibility, and conformational characteristics [33]. The experimental methods consist basically of the synthesis of peptides of a given length, 6-10 amino acids, immobilized on derivatized paper or plastic. These peptides cover the entire linear sequence with overlaps of 2 to 3 amino acids. Next, the peptides that react with appropriate antibodies— polyclonal antibodies derived from infection or from immunization with the native protein—are identified by immunochemical techniques [36].

These methods identify linear epitopes. These should be hard to find because most epitopes are conformational (structural), involving regions that are far apart in the primary sequence, but close to each other in the secondary or tertiary structure of the protein [37]. However, this assertion leads to controversy, as in fact it is not difficult to find linear peptides that constitute an epitope; some authors have therefore said that the different methods for finding and/or defining epitopes are suspect [38,39].

Random peptide generation may be carried out by chemical synthesis [40] as well as by molecular biology techniques [41,42]. Peptides are selected using antibodies against the native protein, whether raised by infection or monoclonal. In these peptide epitopes, the residues and the interactions involved with the antibody may not necessarily be present in the protein; rather, they are mimicking the epitope and are therefore called mimotopes. These methods offer the advantage of being able to simulate conformational epitopes with mimotopes.

B. Improvement of Molecular Orientation and Bond Stability by Covalent Immobilization

As discussed previously, immobilizing a peptide by physical adsorption is very inefficient, since the peptide structure is wholly or partially "frozen" on the solid surface, making reaction with the antibody improbable (Fig. 5). In order to avoid these problems with proteins as well as with peptides, methods have been developed to synthesize nanoparticles with functional groups on their surface, which make it possible to immobilize molecules more precisely by means of covalent bonding. The aims are: (1) to establish bonds that are more stable with respect to time and (2) to orient the peptide precisely so as to favor recognition by the antibody.

When using covalent bonding, contradictions may arise between the selected peptide's reactivity in solution and its reactivity in the immunoassay, since the immobilization process may restrict the peptide from adopting conformations that are important for its recognition—as frequently happens with physical ad-

FIG. 5 Physical adsorption is not the method of choice for peptide immobilization. Attachment to the surface should be controlled so as not to impair peptide flexibility and exposure to the aqueous phase (a). Physical adsorption usually involves multiple interaction points that yield constrained structures (b).

FIG. 5 Physical adsorption is not the method of choice for peptide immobilization. Attachment to the surface should be controlled so as not to impair peptide flexibility and exposure to the aqueous phase (a). Physical adsorption usually involves multiple interaction points that yield constrained structures (b).

sorption. This does not mean that a peptide always loses antigenicity when covalently immobilized; on the contrary, reduction of mobility in a given region of the peptide may enhance recognition. Therefore, the antigenicity of a peptide can only be determined operationally, with explicit reference to the experimental conditions under which it is measured [43]. Flexibility is also important for these small molecules because it determines their ability to adopt appropriate complementarity with the paratope [44]; such flexibility will be possible if the peptides are correctly oriented to the aqueous phase and/or their interactions with the solid phase are minimized.

1. Modification of Peptide Synthesis to Optimize Its Covalent Immobilization

The selected peptide can be modified during synthesis by adding or modifying amino acids (1) in order to achieve efficient immobilization in terms of reaction yield as well as of the final immunoreactivity of the immobilized peptide (Fig. 6); (2) since frequently linear peptides, free from their framework within the native protein, do not adopt the same conformation, it may be necessary to increase conformational similarity between the peptide epitope and the corresponding region of the native protein in order to promote binding of the peptide with the antiprotein antibody.

When designing modifications in order to improve the orientation and exposure of the peptide, the following factors should be taken into consideration: the proper orientation of the peptides on the surface; their separation from the surface and their freedom to rotate around their point of anchorage; a satisfactory immobilization reaction yield; and the possibility of blocking some terminal or o.

added amino acids

FIG. 6 Sequence modifications of the peptides are usually needed to improve immobilization. A common practice is to add amino acids (black line) to the end where covalent immobilization will take place so as to allow conventional conjugation chemistry and improve exposure as well as flexibility.

lateral functional groups, such as thiol, amine, or carboxyl groups, in order to obtain a charge distribution similar to that of the original protein segment.

With respect to how the peptides should be oriented on the surface, which essentially means which end should be used for attachment, some authors give some guidelines for general practice [45]. If the peptide is from the carboxy terminal or amino terminal of the protein, where flexible linear epitopes are frequently found, it is relatively simple to simulate those situations upon immobilization. If the peptide is from the carboxy terminal end of the protein, it should be joined to the surface by its amino end, and vice versa. If the peptide contains a cysteine residue in midsequence, this will be an appropriate anchorage site, and particularly specific in the case of this unique bond, since it is likely that in the original protein this residue forms a disulfide bridge with another part of the protein, and this information may well be available.

When considering giving the peptide freedom to rotate on its axis (Fig. 7), and distance from the surface, the aim is sterically to facilitate its interaction with the antibody while at the same time avoiding interaction with the surface (Fig. 8). Generally, the addition of various glycine residues at the attachment end of the peptide will achieve these aims, as glycine has no lateral side chains to hinder rotation, and if several glycines are used the rest of the peptide will be distanced from the surface.

As will be seen below, certain strategies for making large peptide complexes require the addition of charged amino acids to the attachment end, so as to create a specific "charge cluster." This helps the peptide to diffuse to the molecule to which it will be attached, which bears opposite charges, and also favors a given

FIG. 7 Sequence modifications of the peptides may favor antibody reaction. Amino acids added to the attachment end (black line) will also provide free rotation and movement of the peptide upon its axis (arrows), facilitating antibody recognition.

FIG. 8 Glycine tags as spacer sequences. When peptides are attached directly to the surface, additional undesirable interactions might occur (a). The inclusion of several glycine residues at the attachment end is a good strategy to avoid these interactions (b) and usually does not contribute to nonspecific antibody recognition.

orientation upon immobilization (Fig. 9). Such a strategy also increases the reaction efficiency.

Synthesis modifications to simulate the structure of the peptide within the original protein can be carried out as additional chemical reactions in order to give the peptide a cyclic structure [46], or to add on to either side of the epitope amino acids that are not involved in its reactivity but that can induce a particular conformation [47]. This is especially important in the case of "hairpin" structures [48], which are closely associated with protein antigenicity [49].

2. Methods of Covalent Immobilization onto Particles

Covalent immobilization of the peptide can be carried out in three ways: (1) covalent reaction onto reactive surfaces; (2) forming a macromolecular complex

peptide

FIG. 9 A charged tag or cluster can also be added to the end of the peptide. This procedure may drive an optimal orientation of the peptide, thus improving a covalent immobilization process.

with the peptide, followed by physical adsorption or covalent linkage to the surface; and (3) covering the surface by physical adsorption of polymers possessing reactive groups, followed by covalent immobilization of the peptide.

During covalent reaction processes, it is difficult to avoid physical adsorption onto the surface; therefore, the characteristics of the nonreactive part of the surface are very important. The more hydrophilic it is, the less physical adsorption will occur. Some authors have included inert detergents in their reactions in order to minimize physical adsorption. Several authors have developed special synthesis methods in order to ensure very hydrophilic surfaces for these reactions [50].

(a) Reactive Surfaces. Use of reactive surfaces, or latex particles with added functional groups, is an alternative strategy that bypasses the problems of physical adsorption (Fig. 10). Particles with functional groups are obtained by a process of synthesis in which monomers that produce the base polymer are copoly-merized with other monomers that provide the functional groups. These groups are on the surface of the particles, either in reactive form or in a protected form if they are unstable upon storage.

The first latexes with added functional groups were designed to form cova-lent bonds with the e-amino groups of lysine, present in almost all known proteins. Among the earliest reactive groups used were carboxyl [51], aldehyde [52,53], and chloromethyl groups [54]. At present, other functional groups can be used in latex particle preparation, including amino [55,56], thiol, and epoxy [57] groups, among others (Table 1).

reactive ¡f surface functional group

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