Rabbit antibodies are known for their high affinity and diversity, making them a useful source of research reagents for use in assays. Historically, rabbit monoclonal antibodies were produced using hybridoma technology; however, this is currently driven by more robust single B cell culture and molecular cloning methods.
（Single B-cell rabbit monoclonal antibody development process）
To understand the development of single B-cell antibodies, we will use a customer development project as a case study for your reference: the process of developing a blocking neutralizing rabbit monoclonal antibody based on the single B-cell antibody development platform (for privacy reasons, detailed information related to target A will not be disclosed).
Phase 1: Antigen preparation
The antigen target A of this project is a secreted extracellular protein with a clear receptor protein B. Considering the need for subsequent activity quality control testing of recombinant protein A and subsequent blocking detection and screening of neutralizing antibodies, a eukaryotic mammalian cell recombinant expression system was selected to express the target A protein and its receptor protein B separately, and interaction detection and verification were performed based on ELISA.
（Target A and receptor B protein SDS-PAGE & Elisa interaction testing data）
Phase II: Immunity and Positive B Cell Screening
Rabbits are immunized with the target A antigen recombinant protein obtained in the first phase, including the immunization route, adjuvant, and booster strategy. After the third and fourth immunizations, rabbit serum is analyzed (serum will be evaluated based on antigen ELISA) to identify responding animals . PBMCs and spleens from confirmed positive responding rabbits are then used for differential screening, enrichment, and culture of B cells. The culture supernatants are then validated by ELISA to identify positive B cell clones, which are then screened for neutralizing antibody clones using competitive ELISA.
(Partial data from target A-positive B cell ELISA assay and neutralizing antibody competitive ELISA screening)
Phase III: Sequence and structural analysis of positive single B cell antibodies
Based on the results of the second-stage single B cell culture supernatant test, 25 optimal neutralizing antibody clones were selected to complete antibody gene sequence amplification and sequencing for analysis.
(25 positive antibody variable region sequence frameworks for target A)
The obtained antibody variable region sequence information is used to construct an antibody structural model and predict the binding model between the antibody and target A. Based on the interaction model between target A and its receptor B and the comprehensive detection data of stage 3, suitable clones are selected.
(Left: Interaction model of target A and its receptor B; Right: Binding model of positive cloned antibody and target A)
Phase 4: Antibody recombinant expression and identification
One clone was selected from 25 preferred neutralizing antibody clones for recombinant expression and secondary neutralization test verification.
(Recombinant expression and verification of neutralizing antibodies against target A)
Summary:
At this point, a neutralizing recombinant rabbit monoclonal antibody targeting target A was developed and delivered.
Our R&D team has more than ten years of experience in recombinant protein and recombinant antibody research and development, focusing on eukaryotic mammalian cell recombinant protein expression and single B cell recombinant rabbit monoclonal antibody development, providing simpler and more efficient recombinant protein and recombinant antibody development services.
