A

FIG. 2 Physical adsorption may involve several "interaction points." The flexibility of the molecule favors interaction through multiple points. However, damage of the tertiary and/or quaternary structure of the protein increases with the number of such interactions. Conformational epitopes are very sensitive to denaturation or loss of the three-dimensional structure. After immobilization, the conformational epitope can be (a) preserved, so that antigen-antibody reaction is allowed, or (b) lost, so that antigen-antibody reaction is avoided.

In addition to the problems resulting from a decrease or annulment of recognition because of denaturation on the surface, there may be other problems independent of immunoreactivity. Denaturing particularly affects conforma-tional epitopes, those in which the immunoglobulin recognizes fragments of the macromolecule that are separated in its linear sequence, but close together in the folded molecule. Depending on whether epitopes are mostly linear or confor-mational, denaturing will have a greater or lesser effect on final reactivity. Even if recognition is not affected, the immobilized and denatured protein molecule is a source of nonspecific signals, since nonspecific protein-protein interactions may occur, particularly by means of hydrophobic regions that have been exposed by denaturation, quite independently of the antigen-antibody reaction. Such interactions occurring with proteins present in the sample will contribute to the nonspecificity of the assay. Again, particle agglutination techniques must achieve optimal colloid stability, and self-agglutination caused by denaturation is one of the most serious problems to appear frequently in their development. The denatured protein on the particle surface interacts with that on other particles in a manner similar to that described above.

In the case of antibodies, it has been shown that less than 5% of the immobi-

FIG. 3 Denaturation depends on the available area per protein molecule. Panel A illustrates that tight packaging of the molecules on the surface prevents denaturation, especially for flexible molecules (a), while loose packaging favors it (b). Panels B and C show a schematic representation of the effect of protein concentration—low and high, respectively—during immobilization.

FIG. 3 Denaturation depends on the available area per protein molecule. Panel A illustrates that tight packaging of the molecules on the surface prevents denaturation, especially for flexible molecules (a), while loose packaging favors it (b). Panels B and C show a schematic representation of the effect of protein concentration—low and high, respectively—during immobilization.

lized protein is active [19] in the sense of being capable of recognizing antigen. This low activity may be ascribed as much to immobilization in a way that sterically hinders access to the molecule's paratopes as to modification or dena-turation of the recognition site by the immobilization process.

In any immunoassay—especially those that are dependent on the stability of a colloid—it is important to measure precisely the quantity of bound immunore-agent per unit area. With this figure, important parameters such as the limit of detection and the specificity of the reagent can be modulated. Therefore, all denaturation processes undermine the rational development of new reagents and also often result in the waste of expensive materials.

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