Antibody selectivity

Figure 11 Structure of the phenoxybenzoic acid (PBA) immunogen hapten. Conjugation to the protein through the aldehyde resulted in an immunogen that generated antibodies selective and sensitive for PBA...

Antibodies produced by this procedure were screened for their ability to react with the hapten to form the vinylogous amide 6, which has a convenient UV chromophore near 318nm, clear of the main protein absorption. Two antibodies selected in this way catalysed the expected aldol reaction of acetone with aldehyde 7 by way of the enamine 8 (Scheme 3) the remainder did not. These two effective aldolase mimics have been studied in some detail, and a crystal structure is available for (a Fab fragment of) one of them.126,281... 

This approach has long been used in flow cytometry. The first effort to use secondary antibodies selectively recognizing the corresponding isotype for double... 

Figure 10.3. Schematic representation of monoclonal antibody production using immortalized hybrid cells that secrete antibodies selective for the target antigen. The mortal, immune B cells Isolated from mice immunized with a target antigen are fused with myeloma, immortal B cells that express defective antibodies. The selecting of antigen-specific, immortal hybrid cells (hybridomas) results in identification of unique clones of cells that express antibodies with high specificity and affinity (monoclonal antibodies). These cells are cloned and expanded for large-scale monoclonal antibody preparations.

Parmley, S and Smith, G (1988) Antibody-selectable filamentous fd phage vectors affinity-purification of target genes Gene 73, 305—318. 

Marcus et al. reported that anti GM1 ganglioside antibody can be used as a T cell marker (11, 12, 13). In collaboration with Tada s group of the University of Tokyo we very recently found that antibody to asialoganglio-side GM1 specifically labels mouse natural killer cells and that the antibody selectively kills the cells in the presence of complement (14). Natural killer cells are known by their specific ability to attack tumor cells. We also have evidence that certain gangliosides are capable of eliciting autoimmune diseases experimentally which we called the ganglioside syndrome (JJ5). For example, some brain gangliosides produce autoimmune lesions in peripheral nerve in susceptible rabbits. This may correspond to the human... 

Hawlisch, H., Muller, M., Frank, R., Bautsch, W., Klos, A., and Kohl, J. (2001) Sitespecific anti-C3a receptor single-chain antibodies selected by differential panning on cellulose sheets. Anal. Biochem. 293, 142-145. 

The influence of antibody selection was also described in the work of Niemeyer et al. [37], in which an indirect sandwich assay (Fig. 3D) with a nonfunctionalized primary detection antibody was compared to a direct sandwich assay (Fig. 3C). The removal of one incubation step resulting from the functionalized primary antibody increased coupling efficiency, thereby improving the signal-to-background ratio and, ultimately, the sensitivity of the assay. 

Recent studies show that nucleosome/antinucleosome immune complexes contribute more to lupus nephritis. Serum anti-dsDNA reactivity is always associated with antinucleosome reactivity (A18, B26, B28, B29, C9). Even the highly purified monoclonal and polyclonal anti-dsDNA antibodies selected by affinity chromatography bind to isolated dsDNA and also to nucleosomes (C9, L23). Hybridoma-secreting anti-DNA can also form immune complexes in vitro with nucleosomes released from dying hybridomas in culture (F9). Finally, the binding of an anti-DNA antibody to a nucleosome may render the immune complex more positive and thereby make it more prone to bind to the GBM (T2). 

In an attempt to overcome this problem Kohler and Milstein (1975, 1976) have reported the results of fusing a mouse myeloma cell line with spleen cells taken from a mouse immunised with sheep erythrocytes. The hybrids were cloned and clones secreting anti-sheep erythrocyte antibodies selected. 

The affinity of antibodies selected from naive, synthetic, or even immune phage libraries is typically sufficient for use as a research reagent, but too low for some specific therapeutic applications such as viral neutralization or tumor imaging. For many applications, affinity maturation can be bypassed completely by the construction of multivalent molecules. If this is not sufficient selected antibody clones may be affinity matured (see Figure 18.13). 

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