Paratopes

FIG. 4.1. Schematic representation of two antibodies reacting with a continuous and a discontinuous epitope of a protein antigen interacting residues are indicated in black. If the individual loops of a discontinuous epitope are able to bind to the antibody paratope on their own, they may be given the status of continuous epitope. The inset shows the three loops of an antibody VH chain which form part of the paratope... 

Another difficulty is that a peptide may be able to bind to an existing neutralizing monoclonal antibody by an induced-fit mechanism that is somehow driven by the pre-existing structure of the antibody paratope. However, the same induced-fit process may not take place when the peptide is used as the immunogen and is confronted in the host by a large population of B cell receptors allowing a variety of other interactions. 

Whereas molecular design is a strategy applicable to the chemical level of epitope-paratope interactions, it cannot be used for optimizing the many cellular interactions required for achieving an immune response that leads to infectivity neutralization of a pathogen. As a result, the future development of vaccines will continue to rely more on the empirical testing of the protection afforded by candidate vaccine preparations than on the rational design of biomolecules defined in a reductionist manner by their chemical structure. 

Dilute the resultant complexes in staining buffer containing 10% normal mouse serum to give the working concentration of 5 pg/ml of primary mouse IgG, and incubate for 10 20 min at room temperature to block unbound Fab fragment paratopes. 

Paratope The part of an antibody that recognizes the epitope, i.e., that part of the molecule of an antibody that binds to an antigen. 

A paratope is the complementary portion of the antibody that interacts with an epitope. 

Fig. 12. Noncompetitive hapten immunoassay procedures (A and B) using a combination of the a-type and j6-type anti-idiotype antibodies, each recognizing the framework and paratope of the anti-hapten antibody. Anti-hap, anti-hapten antibody (primary antibody) a-Id, a-type anti-idiotype antibody /J-Id, /i-type anti-idiotype antibody S, signal-generating group B, biotin SA, streptavidin.

Self (S4) first proposed the concept of noncompetitive assay for haptens utilizing an adequate combination of an a-type and a jS-type anti-idiotype antibody, in which he used the term, selective antibody for the a-type antibodies. Then, Barnard and Cohen (Bl) applied this assay principle for the determination of serum E2, naming the assay system an idiometric assay. Figure 12A illustrates the assay procedure of the idiometric assay of E2. The target hapten is captured by excess anti-E2 antibody immobilized on microtiter strips by incubation at room temperature for 1 h (step i). After washing the strips, the /3-type anti-idiotype antibody was added in order to saturate (or block) the unoccupied paratope of the anti-E2 antibody (incubation, room temperature for 30 min) (step ii). The a-type anti-idiotype antibody, which has been labeled with a europium chelate (H4), was then added to the plate and incubated at room temperature for a further 2 h (step iii). Finally, fluorescence intensity due to bound europium was measured with a time-resolved fluorometer. Because of large steric hindrance around the bound jS-type antibody (MW 150,000), the labeled a-type antibody would. 

The use of antibodies has allowed the development of a technique known as immunodiagnostics, especially attractive for the direct analysis of intrinsically complex samples such as blood, serum, urine, food, etc. An immunoassay is a test that uses antibody-antigen complexes as a means of generating a measurable result. The specific interaction between epitopes and paratopes lies at the heart of every immunoassay. The role of antibodies in immunoassays is based on the observation that, in a system containing the determinant and a specific antibody, the distribution of the former between its antibody-bound and antibody-free forms is quantitatively related to the total analyte concentration. 

There are five classes of Igs, or isotypes, in mammals that differ in the sequence of heavy chains and in their carbohydrate contents. The five classes are known as IgG, IgM, IgA, IgD, and IgE and contain different heavy chains known as y, p, ot, 8, and s. In contrast, there are only two types of light chains (X and k) that are the same for all the Ig classes. IgG is the most abundant class of Igs in the serum of mammals and is synthesized predominantly by the plasma cells after secondary exposure to an antigen. They bear two identical paratopes able to bind specifically its complementary antigen [22]. 

Tissue specimens are fixed in formalin and subsequently embedded in paraffin in the majority of cases. While formalin fixation preserves tissue morphology, it also alters the three-dimensional structure of tissue proteins. This alteration can result in a modification of the antigen s epitopes and/or of its electrostatic charges (EC). The loss of an epitope results in an antigen s inability to react with the paratope of the antibody and can only be corrected by the restoration (retrieval) of the epitope. The reduction of net negative ECs of antigens decreases the avidity of the immune reaction and may be compensated for by prolonging the incubation time with the primary antibody (1) or the use of different antibody diluents (2). 

Millar AL, Jackson N, Dalton H, Jennings KR, LeviM,Wahren B, Dimmeck N (1998) Rapid analysis of epitope-paratope interactions between HIV-1 and 17 amino-acid neutralizing microantibody by ESI mass spectrometry. Eur J Biochem 258 164-169... 

The structure of foot-and-mouth disease virus (FMDV) (Acharya et al, 1989) demonstrated that surface receptor interactions could use a quite different mode of attachment. This was developed further by visualizing FMDV-receptor interactions crystallographically (Fry et al, 1999b). Furthermore, in the case of the binding of antibodies by viruses, in the single case for which the crystal structure of a virus-antibody complex has been accomplished, residues from the antibody paratope were found inserted into the canyon (Smith et al, 1996). Thus, although residues within the canyon are more conserved than those outside, and selected... 

These studies are the only structural studies to suggest that antibody binding may affect the capsid in ways other than just small, localized changes at the epitope—paratope interface. However, these changes are not related to bivalent binding of the antibody, nor are they related to neutralization efficacy. The conclusion from these studies is, again, that the major effect of the antibody on viral infectivity is steric abrogation of receptor-virus interactions. 

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