Construction of Immunized Rabbit Antibody Library

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 are the greatest challenge in antibody library construction that are 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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