To identify molecules of interest in research and diagnostic settings, scientists can use antibodies produced by animal immune systems to bind precisely to antigens as a starting point for manufacturing probes. No other technology currently available enables scientists to develop and produce tools for molecular recognition that are as highly specialized as antibodies.
Most medical or cell biology researchers who conduct any molecular testing employ antibody technology in some capacity. Different researchers will spend more or less time on antibody synthesis and purification, using other hosts depending on their demands.
During the 1970s and 1980s, antibody production methods were created for making, purifying, and changing antibodies for antigen-specific probes. These methods have mostly stayed the same since Harlow and Lane published their classic Antibodies: A Laboratory Manual in 1988.
However, animal hosts have also risen in popularity over the past years.
This guide discusses antibody production in different hosts, especially rabbits and mice.
The Rising Popularity of Rabbit Hosts vs. Mouse Hosts
Since the hybridoma joined other antibody production techniques after it was first discovered in the 1970s, mice have become the primary host systems for synthesizing antibodies. However, as researchers have extended our understanding of the immune systems of various animals, other hosts have garnered interest thanks to their distinctive qualities, as opposed tomouse as a host for antibody production.
Scientists first became interested in these hosts when they found out rabbits could be viable alternatives. In contrast to rodents' immune systems, rabbits appeared to be able to identify a significantly wider variety of antigens. Surprisingly, the products of rabbit antibody production have a much higher affinity, particularly for epitopes that come from humans or epitopes identified as non-immunogenic in mice.
These qualities, coupled with rabbits' bigger size than mice's, have rabbit as a host for antibody production, particularly efficient for study and diagnosis.
Antibody Production in Rabbits and Immune Reaction
Like the immune reaction in humans, the production of antibodies in rabbits is caused by the intricate relationship of antigen-presenting cells (APC), T cells, and B cells, which then change into plasma cells that make antibodies.
The first immune reaction causes the rabbit's body to make the IgM isotype. This is followed by a release of IgG (up to 20 mg/ml) or IgA (up to 4 mg/ml) afterward. So far, the IgD isotype in rabbits has yet to be matched up with anything else. In contrast to other species, rabbits' IgG isotype does not show subclass differentiation.
The rabbit antibody shares structural similarities with its mammalian and human counterparts, but their components differ. For example, rabbit IgG's N terminus and the D-E loop tend to have fewer amino acid residues. In addition, these biomolecules have a peculiar interdomain disulfide bond in their structure. Researchers speculate that this link contributes significantly to making these molecules more stable and extending their shelf lives.
In addition, it was discovered that the CDR3-loop, the third complementarity-determining area of the light chain, was noticeably lengthier in rabbits compared to its length in humans and mice. The higher binding power of these bioreagents is also attributed to this property.
In addition to the structural variations, rabbits' methods for developing their antibody repertoires are notably distinct from those documented for rodents and humans. Also, the rabbit's size and the conditions in which it grows considerably impact its varied naive repertory. For instance, newborn bunnies have a weakened immune system compared to older rabbits.
Additionally, comparative antibody production in rabbits brought up in germ-free environments has been found to produce a primary antibody repertoire that is only moderately diverse.
In contrast to humans and rodents, rabbits have a substantially smaller VH repertory, although their VL repertoire is considerably more extensive and varied. Although these hosts have a smaller VH repertoire, they often make up for it with much higher rates of gene conversion and somatic hypermutation.
Production of Polyclonal Antibodies in Rabbits as Opposed to Mice
In most cases, rabbit polyclonal antibodies are chosen above their rat counterparts for use in diagnostics and in writing analytical essays. This preference can be traced back to three primary causes, the first of which is associated with the physical size of rabbits. These hosts can produce significantly more antisera because they are far more significant than the ordinary rats employed in the manufacture of antibodies.
In addition, rabbits can produce antibodies opposing antigens that typically do not cause a reaction from the immune system of rodents. Finally, rabbits are effective at producing antisera with a high titer of antibodies and greater sensitivity and affinity towards a particular target. This is backed by extensive research.
Rabbit Monoclonal Antibody Production
Using Hybrid Technology
There is still a significant need for polyclonal rabbit antibodies as reagents. But, just like traditional polyclonal antibodies made from rodents, these products vary from batch to batch. As a result, monoclonal antibodies should be utilized instead of polyclonal antibodies for applications requiring high uniformity and repeatability.
Generating rabbit monoclonal antibodies is substantially more challenging than generating their mouse equivalents. Hybridoma technology was initially used to try and produce these antibodies. However, these efforts were unsuccessful because rabbit spleen cells did not have access to a suitable fusion partner at the time.
Numerous studies were conducted on the early hybrid cell lines of mouse-rabbit heterohybridomas. However, it was discovered that they were prone to instability and were challenging to clone. A study conducted in 1995 at Loyola University in Chicago (United States) and directed by Katherine Knight was responsible for the first significant advance in the field.
The researchers used a double-transgenic rabbit that overexpressed the v-abl and c-myc oncogenes to create a compatible fusion partner for rabbit spleen cells. The transgenic rabbit got a growth that looked like a myeloma, which made it possible to separate the 240E-1 cell line.
After that, researchers fused the 240E-1 cell line with cells from rabbit spleens. However, the resulting hybridomas were genetically prone to instability and rapidly losing the genes that encoded antibodies. Weimin Zhu and Robert Pytela, scientists at the University of California, San Francisco (USA), eventually found solutions to the stability issues. These scientists chose to subclone 240E-1 multiple times and optimize the medium to stabilize it.