Preferential rabbit antibody responses to C-termini of NOTCH3 peptide immunogens.

Antibodies raised in peptide-immunized rabbits have been used in biological research for decades. Although there has been wide implementation of this approach, specific proteins are occasionally difficult to target for multiple reasons. One consideration that was noted in mice is that humoral responses may preferentially target the carboxyl terminus of the peptide sequence which is not present in the intact protein. To shed light on the frequency of preferential rabbit antibody responses to C-termini of peptide immunogens, we present our experience with generation of rabbit antibodies to human NOTCH3. A total of 23 antibodies were raised against 10 peptide sequences of human NOTCH3. Over 70% (16 of 23) of these polyclonal antibodies were determined to be C-terminal preferring: NOTCH3 peptide-reactive antibodies largely targeted the terminating free carboxyl group of the immunizing peptide. The antibodies that preferred C-terminal epitopes reacted weakly or not at all with recombinant target sequences with extension the C-terminus that eliminated the free carboxyl group of the immunogen structure; furthermore, each of these antisera revealed no antibody reactivity to proteins truncated before the C-terminus of the immunogen. In immunocytochemical applications of these anti-peptide antibodies, we similarly found reactivity to recombinant targets that best binding to cells expressing the free C-terminus of the immunizing sequence. In aggregate, our experience demonstrates a strong propensity for rabbits to mount antibody responses to C-terminal epitopes of NOTCH3-derived peptides which is predicted to limit their use against the native protein. We discuss some potential approaches to overcome this bias that could improve the efficiency of generation of antibodies in this commonly utilized experimental paradigm.

May the Odds Be Ever in Your Favor: Non-deterministic Mechanisms Diversifying Cell Surface Molecule Expression.

How does the information in the genome program the functions of the wide variety of cells in the body? While the development of biological organisms appears to follow an explicit set of genomic instructions to generate the same outcome each time, many biological mechanisms harness molecular noise to produce variable outcomes. Non-deterministic variation is frequently observed in the diversification of cell surface molecules that give cells their functional properties, and is observed across eukaryotic clades, from single-celled protozoans to mammals. This is particularly evident in immune systems, where random recombination produces millions of antibodies from only a few genes; in nervous systems, where stochastic mechanisms vary the sensory receptors and synaptic matching molecules produced by different neurons; and in microbial antigenic variation. These systems employ overlapping molecular strategies including allelic exclusion, gene silencing by constitutive heterochromatin, targeted double-strand breaks, and competition for limiting enhancers. Here, we describe and compare five stochastic molecular mechanisms that produce variety in pathogen coat proteins and in the cell surface receptors of animal immune and neuronal cells, with an emphasis on the utility of non-deterministic variation.