Antibodies to small molecules

For small molecules, the immune response is often small or nonexistent [the immune system has evolved to deal with larger invaders such as bacteria]. However, the scientist can still use the immune system of a laboratory animal to produce antibodies to small molecule analytes (S) he need only first chemically couple the analyte to a larger molecule, a carrier protein. The analyte is now known as a hapten (Fig. 2). Only a minority of the antibodies produced may be selective for the hapten, rather than the carrier protein, but the number still usually exceeds that which can be obtained by injecting the analyte alone [13]. 

The ability of a molecule to elicit an immune response is in part linked to its size. Few molecules with a molecular weight < 2000 are immunogenic. However, antibodies to small molecules (haptens) can be produced if the hapten is chemically linked to a larger carrier molecule (e.g. bovine serum albumin), and the hapten carrier molecule complex used as an immunogen. 

The concept of immunoassay was first described in 1945 when Landsteiner suggested that antibodies could bind selectively to small molecules (haptens) when they were conjugated to a larger carrier molecule. This hapten-specific concept was explored by Yalow and Berson in the late 1950s, and resulted in an immunoassay that was applied to insulin monitoring in humans. This pioneering work set the stage for the rapid advancement of immunochemical methods for clinical use. 

Rather than using the protein molecule as a whole, the imprinting of selected protein epitopes may present a more practical approach. Imprints of such patches may then act as receptors for these parts of the protein. It could be shown that an MIP imprinted with a tetrapeptide was able to recognize not only the template but also a protein bearing the same 3-amino acid terminus as the peptide template [129]. If this approach proves to be successful in other cases as well, MI-based recognition will no longer be limited to small molecules. The result will be even more antibody like biomimetic polymers. 

The selection considerations for appropriate p7 markers for cIEF with proteins/anti bodies included purity and stability of the p7 markers, p7 values of the protein analytes, and potential protein—p7 marker interactions. High purity, stable p7 markers that give reliable p7 values with no protein—p7 marker interaction are desirable. Table 6 lists sets of p7 markers used for optimization. The antibody of interest had a p7 range of approximately 6.3 to 7.0. In this case, six different vendor sources were evaluated. These p7 markers vary in nature, from proteins and peptides to small molecules. The e-grams obtained using these markers with the antibody of interest are shown in Figure 22. Although the nature of the p7 markers and exact p7 marker values were different, the cIFF profiles of the antibody were the same. 

The production of antibodies for small molecules is difficult to achieve as they must be coupled to a carrier protein before inoculation. Moreover, such conjugation may change the structural properties of the antigen exposed to the immune system and the obtained antibodies will be directed against a structure slightly different from the target compound. 

Most of the recently developed methods for the detection, characterization, and quantitation of proteins are immunoassays based on the fact that proteins are antigens, compounds that can be recognized by an antibody. It is also true that by combining small molecules (haptens) with a larger carrier molecule such as a protein, these methods can be extended to small molecules of interest since antibodies can be produced that recognize epitopes (specific sites on the antigen recognized by the antibody) that include the hapten. 

Compared to small-molecule dmgs, therapeutic mAbs display different pharmacokinetic characteristics, including nonlinear pharmacokinetic behavior. As the majority of therapeutic mAbs present IgG (or more specially IgGl) molecules, the emphasis will be placed on this isotype, although the characteristics of newer types of molecule such as antibody fragments will also be included. 

To date, no studies on the metabolism of cetuximab have been performed in humans or in animals. Indeed, metabolism studies are not generally performed for mAbs. Several pathways have been described that may contribute to antibody metabolism, all of which involve biodegradation of the antibody to smaller molecules (i. e., small peptides or amino acids). This fact has been recognized in the International Conference on Harmonization (ICH) guidance document Preclinical Safety Evaluation of Biotechnology-Derived Pharmaceuticals [22], where it is stated in Section 4.2.3 that "... the expected consequence of metabolism of biotechnology-derived pharmaceuticals is the degradation to small peptides and individual amino acids. .. and that therefore classical biotransformation studies as performed for traditional small molecule pharmaceuticals are not needed. 

This method is used to test the effect of such compounds as soluble CD4 that bind to gpl20 expressed on the surface of infected cells. Similarly, this system could be used to investigate the antibodies or small molecules that may bind to the family of coreceptors (CXR4 and CCR5, and so forth) required for the entry of HIV into cells. Compounds that selectively kill HIV-infected cells may also be investigated with this technique (6). If CPE or cell viability (vital staining) is used as an end-point then plates should be treated as described in Note 6. 

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