Monoclonal antibody formats

Debaene, F., Wagner-Rousset, E., Colas, O., Ayoub, D., Corvaia, N., Van Dorsse-laer. A., Beck, A., Cianferani, S. (2013) Time Resolved Native Ion-Mobility Mass Spectrometry to Monitor Dynamics of IgG4 Fab Arm Exchange and Bispecific Monoclonal Antibody Formation. Anal. Chem. 85 9785-9792. 

Product formation kinetics in mammalian cells has been studied extensively for hybridomas. Most monoclonal antibodies are produced at an enhanced rate during the Gq phase of the cell cycle (8—10). A model for antibody production based on this cell cycle dependence and traditional Monod kinetics for cell growth has been proposed (11). However, it is not clear if this cell cycle dependence carries over to recombinant CHO cells. In fact it has been reported that dihydrofolate reductase, the gene for which is co-amplified with the gene for the recombinant protein in CHO cells, synthesis is associated with the S phase of the cell cycle (12). Hence it is possible that the product formation kinetics in recombinant CHO cells is different from that of hybridomas. 

In cultures of the more resistant cell line, MDCK, the predominant effect of Tyv-specific monoclonal antibodies is to exclude larvae from the monolayer. This exclusion is associated with the formation of cap-like structures that cover the stoma of the larva. Such caps were first described... 

The ratio of primary antibodies to Fab fragments required for the formation of complexes that produce optimal immunolabeling of specific antigen does not appear to vary dramatically with primary antibody specificity or species. Primary antibody to Fab fragment ratios of 1 2 1 4 (weight for weight, based on concentration data supplied by manufacturers) typically produces optimal results with primary mouse monoclonal antibodies. 

Enzyme labels are usually associated with solid-phase antibodies in the technique known as enzyme-linked immunosorbent assay (ELISA). There are several variants of this technique employing both competitive and non-competitive systems. However it is best used in combination with two monoclonal antibodies in the two-site format in which an excess of antibody is bound to a solid phase such as a test-tube or microtitre plate the test antigen is then added and is largely sequestered by the antibody (Figure 7.12). After washing... 

The most commonly used format for quantitation assays is the sandwich assay format. Typically, a monoclonal antibody (MAb) is used to capture the product. It is then detected by another antibody, usually enzyme-labeled. A reference standard is used from which to compare the response of an unknown test sample. There is a relative increase in measured response (optical density) with increasing analyte concentration. Figure 11.2 is an example of a typical ELISA standard curve. 

Tanaka, H., Fukuda, N., and Shoyama, Y. (1999). Formation of monoclonal antibody against a major ginseng component, ginsenoside Rbl and its characterization. Cytotechnology 29, 115-120. 

The process of transcytosis is illustrated in Figure 2.3 for the transferrin receptor (TfR) [37]. The receptor is heavily expressed at the BBB compared to other vascular beds [38]. Transferrin or a monoclonal antibody to the extracellular domain of the receptor protein will bind from the luminal side of the BBB. This triggers cellular uptake by the mechanism of receptor-mediated endocytosis, i.e. the invagination and budding off of parts of the cell membrane as a result of the formation of small vesicles (endosomes). The transceUular passage of ligand (transcytosis) is completed by exocytosis at the abluminal membrane, and the whole process is completed within minutes in vivo. 

However, recombinant antibodies may be less stable and have lower binding affinities than monoclonal antibodies (Valle and Jendoubi, 2003). Therefore, in order to fully implement the microarray format, a host of diverse capture agents could be required in addition to antibodies. These include peptides, small molecules, aptamers, ribozymes, or other molecular recognition probes yet to be discovered. However, it is also xmderstandable because of the diverse nature of proteins that additional technologies besides microarrays will be used in proteomics research (Hanash, 2003). 

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