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Construction of Immunized Rabbit Antibody Library
Monoclonal Rabbit Antibody Production
In contrast to rabbit hybridoma technology available in other leading companies, Creative Biolabs has built up a unique rabbit monoclonal antibody production platform based on our unparalleled expertise in phage display antibody library technology. We generate rabbit antibody libraries and select high affinity antigen-specific antibodies by phage display following Dr. Christoph Rader's methodology. Usually, two or more rabbits of the b9 allotype are immunized with one antigen. Total RNA from spleen lymphocytes and bone marrow cells are purified, and then the first-strand cDNAs are synthesized. cDNA from spleen and bone marrow samples derived from multiple groups of animals immunized by different antigens can be pooled to make a unified library for all the antigens. Chimeric rabbit/human Fab libraries are made in our pC Display phagemid vectors by fusing rabbit Vκ/Vλ to human Cκ and rabbit VH to human CH1 of human IgG1.
The rabbit antibody repertoire is an exceptional source for both polyclonal antibodies and monoclonal antibodies that combine high specificity with high avidity and affinity. In addition, rabbits, which belong to the order Lagomorpha (lagomorphs), are evolutionarily distant from mice and rats, which belong to the order Rodentia (rodents). As a consequence, epitopes conserved between rodent and human antigens that are invisible to rodent monoclonal antibodies (and also human monoclonal antibodies generated from transgenic mice with human immunoglobulin genes) can often be recognized by rabbit antibodies. Rabbit monoclonal antibodies have overcome this limitation, providing access to defined reagents of infinite supply from the rabbit antibody repertoire. Rabbit monoclonal antibodies generated by phage display offer additional advantages due to the fact that the phenotype (protein) and genotype (cDNA) are selected at the same time. Also, in comparison with rabbit hybirdoma technology, our phage-display platform can produce much more antibody clones at a time.
It was well-known that rabbit Fab's are toxic to E. coli cells, in which phage display systems work. There are two approved approaches to avoid toxicity, insolubility and low expression problems of rabbit Fab in bacterial cells. The first is to make rabbit/human chimeric Fab libraries in which rabbit variable domains (Vλ, Vk, and VH) are linked with human constant domains (Ck and CH1). This approach has been well demonstrated by the work done by Drs. Carlos F. Barbas III and Christoph Rader. The other approach is to express the selected rabbit Fab (or scFv) antibodies in yeast Pichia pastoris, relying on yeast display technology, which is current not available at our company.
Of note, most rabbit light chains have an extra disulfide bridge that links the variable and constant domains in addition to the two intra-chain disulfide bridges shared with mouse and human κ light chains. By evaluating the impact of this increased disulfide bridge complexity on the generation and selection of chimeric rabbit/human Fab libraries, scientists have demonstrated that rabbits with mutant bas and wild-type parental b9 allotypes are excellent sources for rabbit monoclonal antibodies.
We have constructed a large number of immunized rabbit antibody libraries. In particular, we developed an in-frame-selection technique to exclude Fab library clones that have shortened antibody fragments and reading frame shifts, which is the greatest challenge in antibody library construction that is usually due to the errors in synthetic PCR primers and PCR reactions used to amplify antibody genes.
Screening of Immunized Rabbit Antibody Library
Selection of high affinity binders is mainly based on the in-solution phage display antibody library screening strategies we employ. We have two strategies: selection based on the equilibrium constant (Kd) and selection based on binding kinetics. In the first approach, sub-library phage is incubated with biotinylated antigen at controlled concentrations and bound phages are captured by immobilized NeutrAvidin. The second, selection based on binding kinetics is also termed off-rate (Koff) selection, in which phage population is allowed to saturate the labeled antigen before a large molar excess of unlabeled antigen is added to the mix for controlled periods of time. This allows the selection of mutant antibodies that have slower off-rates. Since a reduction in Koff usually results in a higher affinity, this selection approach singles out antibody variants with improved Kd. These methods allow selection of antibodies that have high affinity. In the end, the selected antibody fragments can be expressed to test their binding specificity and affinity.
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