The rabbit-derived antibodies are well-known for their high affinity and diversity, making them a valuable source for research reagents in detection analysis. In the past, rabbit monoclonal antibodies were produced using hybridoma technology; however, currently, they are driven by more powerful single B-cell culture and molecular cloning methods.
（Single B-cell rabbit monoclonal antibody development process）
For an understanding of single B-cell antibody development, we present a customer development project as a case study: the development of a neutralizing rabbit monoclonal antibody through the single B-cell antibody development platform (specific details about Target A are not disclosed due to privacy reasons).
Stage One: Antigen Preparation
The antigen target A for this project is a secreted extracellular protein with a clear receptor protein B. Considering the later quality control testing for recombinant protein A and the need for screening neutralizing antibodies, a eukaryotic mammalian cell recombinant expression system was chosen. The target A protein and its receptor protein B were expressed separately, and their interaction was verified based on Elisa.
（Target A and receptor B protein SDS-PAGE & Elisa interaction testing data）
Stage Two: Immunization and Positive B-cell Selection
Rabbits were immunized with the recombinant protein of target A obtained in Stage One, including the immunization route, adjuvants, and enhanced immunization strategies. After the third and fourth immunizations, rabbit sera were analyzed (sera were evaluated based on antigen ELISA testing) to determine responsive animals. PBMC and spleen were obtained from the identified positive response rabbits for differentiated B-cell screening, enrichment, and cultivation. The culture supernatant was then tested using Elisa to validate positive B-cell clones, followed by competitive Elisa testing for selecting neutralizing antibody clones.
（Target A positive B-cell Elisa testing and neutralizing antibody competitive Elisa screening partial data）
Stage Three: Positive Single B-cell Antibody Sequencing and Structural Analysis
Based on the results of the second-stage single B-cell culture supernatant testing, 25 preferred neutralizing antibody clones were selected for antibody gene sequence amplification and subsequent sequencing and analysis.
（Target A's 25 positive antibody variable region sequence frameworks）
The obtained antibody variable region sequence information was used to construct an antibody structure model and predict the binding model of the antibody and Target A. This was based on the interaction model between Target A and its receptor protein B. Considering the data from Stage Three, a suitable clone was selected.
（Left: Interaction model between Target A and its receptor B; Right: Binding model of positive clone antibody with Target A）
Stage Four: Antibody Recombinant Expression and Identification
One clone was selected from the 25 preferred neutralizing antibody clones for recombinant expression, followed by secondary neutralization testing validation.
（Target A neutralizing antibody recombinant expression testing validation）
In conclusion, the development and delivery of a recombinant rabbit monoclonal antibody targeting Target A with neutralizing properties have been completed.
Our R&D team has over ten years of experience in recombinant protein and recombinant antibody development, focusing on eukaryotic mammalian cell recombinant protein expression and single B-cell recombinant rabbit monoclonal antibody development, providing a simpler and more efficient service for recombinant protein and recombinant antibody development.
