Creation antibodies

This chapter describes the design, preparation, and use of hapten-carrier conjugates used to elicit an immune response toward a coupled hapten. The chemical reactions discussed for these conjugations are useful for coupling peptides, proteins, carbohydrates, oligonucleotides, and other small organic molecules to various carrier macromolecules. The resultant conjugates are important in antibody production, immune response research, and in the creation of vaccines. 

Sensors are usually attached chemically or physically to other materials here referred as the carrier, like polymers, antibodies, and optical fibers in order to facilitate the sensing process. These carriers generally affect the luminescent characteristics of the sensor molecules. The modification of the luminescent characteristics of the sensor is caused by the creation of more than one microphase or microenvironment for the sensor. Each molecule in its particular microenvironment may return to the ground state following a different set of processes or mechanisms. Alternatively, the nonra-diative decay rate of each microphase may be different for each sensor molecule. Depending on the characteristics of the carrier and the sensor, the number of microphases may be one, two, three, or an infinite number. 

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].

Substrates for the creation of protein microarrays were initially selected from those used for DNA arrays, for example, PLL glass slides. At first, these substrates proved to be sufficient for antibody microarray studies. However, not all proteins will behave well or similarly on a particular substrate material. New solid phases applicable for protein microarrays need to be found. 

Figure 7.20 Examples of haptens for the creation of antibodies directed toward the 6-phosphor-ylserine tumor suppressive protein p53. ...

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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