Creation antibodies with

Figure 11.6. Schematic diagram showing the assembly of IL-12 protein for antibody-based drug delivery, (a) The mature sequences of the p35 subunit of IL-12 are fused to the C-terminus of the heavy chain of a tumour-specific antibody and co-expressed with the antibody light chain and the p40 subunit of IL-12. Formation of the final immunocytokine requires the creation of disulfide bridges between the antibody chains and interactions of p35 and p40 subunits of IL-12 [119]. (b) Alternatively the IgG heavy chain and both subunits of IL-12 can be linked via flexible linkers allowing for equimolar assembly of IL-12 [120].

Molecular recognition, defined as the favored binding of a molecule (i.e., a substrate) to a specific site in a receptor over other structurally and chemically related molecules, is at the forefront of science.1 s Long before man walked on this earth, nature had succeeded in the creation of a series of biologically based recognition elements with unmatched specificity antibodies, enzymes, and receptors. Perhaps the simplest well-known example of this concept is the lock and key hypothesis that has been used to describe protein-substrate interactions in biological systems.5-7... 

The high sample demands and low-throughput of LC-MS methods have led to the creation of a capillary electrophoresis (CE) platform for ABPP [48]. Proteomes are labeled with a fluorescent probe, digested with trypsin, and enriched with antifluorophore antibody resins. Use of CE coupled with laser-induced fluorescence (LIF) detection to analyze the enriched peptides resulted in far superior resolution to ID SDS-PAGE, particularly for enzymes that share similar molecular masses. Sensitivity limits of 0.05-0.1 pmol/mg proteome, negligible sample requirements (—0.01—0.1 pg proteome), and the ability to perform rapid CE runs in parallel with 96-channel instruments, make CE-based ABPP a potentially powerful technique. One drawback is that the identities of the probe-labeled proteins are not immediately apparent, and correlated LC-MS experiments must be performed to assign protein identities to the peaks on the CE readout. 

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