Cell sorting antibodies

Immunoassay kit, where the antibodies of 8-oxoguanine (96) are conjugated with fluorescein isothiocyante (97) as fluorophore and combine with the oxidized DNA. Detection of the greenish fluorescence is by fluorescence microscopy for tissues or by fluorescence-activated cell sorting for cell suspensions236. 

Antibodies labeled with fluorescent molecules have several applications, particularly in cytochemistry and cell sorting. There are many fluorochromes used in labeling (1), such as coumarin derivatives, phycobiliproteins, and rare earth chelates however, fluorescein and rhodamine (Table 1) are the most commonly used. 

Pharmacokinetic data analysis requires determination of the analyte in various body fluids. In the case of therapeutic antibodies, serum is the most common matrix to be analyzed. For a critical interpretation of pharmacokinetic data the chosen bioanalytical methods must be considered. The most frequently used for mAbs include enzyme-linked immunosorbent assay (ELISA), capillary electrophoresis (CE)/polyacrylamide gel electrophoresis (PAGE), fluorescence-activated cell sorting (FACS), and surface plasmon resonance (SPR). The challenges and limitations of bioanalytical methods used for the analysis of mAb concentrations are discussed in detail in Chapter 6. 

Francisco, J. A., Campbell, R., Iverson, B. L., and Georgiou, G. (1993). Production and fluorescence-activated cell sorting of Escherichia coli expressing a functional antibody fragment on the external surface. Proc. Natl. Acad. Sci. USA 90, 10444—10448. 

The isolation of an antibody is an extremely useful first step toward understanding the function of the protein to which it binds. scFvs derived from phage antibody libraries have been used in immunofluorescence, immunoprecipitation, fluorescence-activated cell sorting, Western blotting, and inhibition of function studies, both in vivo, in tissue culture cells, and in vitro. In this sense, they can essentially be used in the same way as conventional hybridoma-derived antibodies. They have the advantage, however, that the genes for the variable regions are cloned simultaneously with selection. This allows the fusion of functional elements, such as dimerization domains, effector or detector functions to selected scFvs [38], the re-creation of complete... 

Note. ELISA = enzyme-linked immunosorbent assay, FACS = fluorescence-activated cell sorting, HPLC = high-performance liquid chromatography, IHC = immunohistochemistry, LC/MS = liquid chromatography/MS = mass spectrometry, LPS = lipopolysaccharide (endotoxin), NA = not applicable, PAHA = (nonhuman) primate anti-human antibodies. 

Bailey RC, et al. DNA-encoded antibody libraries a unified platform for multiplexed cell sorting and detection of genes and proteins. J Am. Chem. Soc. 2007 129 1959-1967. 

The PD behavior of therapeutic MAbs is complex. Fortunately, the activity of these agents can be evaluated by the use of fluorescence activated cell sorting (FACS). Like most therapeutic biologies, the mechanism of action is commonly described using the standard indirect effect models. However, because FACS data can determine the fraction of cell surface receptor bound by the MAb, and because the change in receptor density can be followed using this same method, more mechanistic models have been proposed (29). In these models, the relationship between concentration of antibody and bound receptors can be explicitly described and the bound antibody is then used to drive the indirect effect model. A schematic of this model is provided in Figure 41.10. 

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