Rabbit Monoclonal Antibody Discovery Using DNA Immunization
We are pleased to announce the launch of a new CRO service for rabbit monoclonal antibody development using our proprietary single B-cell screening technique (PAT: WO2024071374). By combining DNA immunization and rabbit monoclonal antibody production, we are able to obtain antibodies that possess both the benefits of the DNA immunization method and the "high efficacy and affinity" properties of monoclonal rabbit antibodies.
What is DNA immunization? Antibody Production Technology
DNA immunization is a method of antibody production. An expression vector containing the cDNA of the target antigen is produced, and the vector is directly administered to the immunized animal.
The cDNA of the target antigen is transcribed by the promoter of the vector and expressed in the body of the immunized animal to acquire immunogenicity.
The DNA immunization method has a high success rate for antibodies against membrane proteins, which are difficult to obtain with conventional immunization using antigens (peptides/proteins).
In the conventional method of producing monoclonal antibodies, recombinant proteins are prepared from DNA information or peptide synthesis, and these are administered as antigens to immunized animals to obtain antibodies. The DNA immunization method, on the other hand, induces immunity by injecting a plasmid directly into the immunized animal and expressing the target protein in the animal's body. Compared to the conventional protein/peptide immunization method, the DNA immunization method has the advantages of bypassing the need for the antigen protein/peptide, while avoiding antigen contamination, antigen denaturation and allowing for easy modification of the target antigen.
Another feature is that neutralizing antibodies (functional antibodies) are easily obtained.
Features of DNA immunization
- No need to prepare purified antigen.
- Antibodies that recognize native form of proteins can be produced.
- Antibodies to multiple transmembrane proteins can be produced.
- Antibodies to highly homologous targets can be produced.
Features of Rabbit Antibodies
- High affinity and specificity compared to mouse/rat
- Antibodies with a diverse epitope repertoire can be obtained.
- High antigen reactivity for antibody production against rare or weak antigens
DNA immunization x Rabbit monoclonal antibody
Rabbit Monoclonal Antibody Production
Rabbit Monoclonal Antibody Production Monoclonal rabbit antibodies have been difficult to produce for technical reasons. In particular, the inefficiency of the hybridoma method, which is the golden standard for monoclonal antibody production, has been a technical hurdle. Instead of using hybridoma technology, we have made it possible to efficiently produce rabbit monoclonal antibodies by introducing our proprietary FUJIFILM Group single B-cell screening technology (PAT: WO2024071374).
Rabbit monoclonal antibody production by DNA immunization
We have more than 20 years of experience in producing antibodies by DNA immunization in rats and mice, and have delivered high quality antibodies to many customers.
By combining DNA immunization and rabbit monoclonal antibody production, we are able to obtain antibodies that possess the benefits of the DNA Immunization method and the "high performance and affinity" properties of monoclonal rabbit antibodies.
These antibodies can be used as a next-generation antibodies for antibody therapeutics (i.e., TCR/CAR-T, ADCs), diagnostic reagents, and more.
Rabbit DNA Immunization Service Flow and Price
Feasibility Study: Planning of Antibody Production Strategy
Based on the requested sequence information and intended use, we analyze the structure and properties of the target in silico and propose an antibody production strategy.
In addition, we will propose the optimal immunizing region, immunization method, and screening method together with a report.
| Milestone Name | Lead Time |
|---|---|
| Feasibility Study (Development of antibody production strategies) | 2 weeks |
Milestone 1: Antigen Expression Analysis in Mammalian Cells
Cell surface expression confirmation: Based on the information determined in the Feasibility Study described above, target genes are cloned into vectors provided by the customer or into our inhouse expression vectors to create immunizing plasmids. After transient expression in mammalian cells, we check antigen expression on the cell surface using a flow cytometer.
If you already have a vector that is known to express the antigen, Milestone 1 can be omitted.
| Milestone Name | Lead Time |
|---|---|
| Analysis of antigen expression in mammalian cells | 1.5 months |
Milestone 2: Rabbit Immunization
Animal immunization: Rabbits are immunized with the immunizing plasmid. Immunization is done subcutaneously, and antiserum is collected after about 2 months of immunization. Antibody titers are evaluated by flow cytometry using transient forced expression cells produced in Milestone 1.
| Milestone Name | Lead Time |
|---|---|
| Rabbit immunization (3 rabbits*) | around 2 months |
*The rabbit species we use are Japanese White rabbits. For other strains, an additional fee will be charged.
Optional specifications (will incur additional fees):
- Additional number of immunized animals (4 or more)
- Booster immunization
- FCM analysis using customer-owned cell lines
- Evaluation by ELISA
(removed options about sending samples due to shipping restrictions of vet commodities)
Milestone 3: Monoclonal antibody production through single B-cell cloning
B-cell cloning: Spleens from rabbits with elevated antibody titers are harvested and splenocytes are obtained. Splenocytes are seeded into plates with approximately 200,000 holes (chambers) and cells are separated one by one.
Screening:
Primary screening is conducted in chambers seeded with spleen cells. By coating the chamber with extracellular vesicles (EVs) that express antigens on their membranes, membrane proteins can be screened according to our Fujifilm proprietary method (PAT: WO2024071374). The chambers are coated with antigen, and cells that secrete antibodies binding to the antigen are picked (up to 44 cells). The antibody genes are then amplified via single-cell PCR. In secondary screening, the amplified gene fragments are transiently expressed in mammalian cells, and their antigen-binding capabilities are assessed using flow cytometry or ELISA. Optionally, we can send the culture supernatant (up to several hundred μL) for evaluation at your facility.
Cloning:
Six clones are selected and an expression vector carrying the antibody gene is constructed. The expression vector is transiently expressed in mammalian cells, and antigen binding and expression levels are analyzed by flow cytometry or ELISA. We will then sequence the antibody genes and deliver the antibody gene sequences and expression vectors.
| Milestone Name | Lead Time |
|---|---|
| Monoclonal antibody production by single B-cell cloning | 2-3 months |
- If less than 20 cells are positive in the primary screening, we will screen additional plates at an added cost.
Optional specifications (will incur additional fees):
- Screening with additional plate (less than 20 positive cells in primary screening)
- Screening with additional plate (more than 20 positive cells in primary screening)
- Additional clones (7 or more clones)
- Additional screening by ELISA (secondary screening)
- Additional screening by ELISA (cloning)
Deliverables
- Antibody gene sequence
- Report
Inquiry
For research use or further manufacturing use only. Not for use in diagnostic procedures.
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