The Hybridoma Core provides investigators at Lerner Research Institute with a resource for monoclonal antibody production. The facility also provides consultation services to assist the investigator in all aspects of monoclonal antibody production. The services are performed under strict quality control guidelines appropriate for antibodies that may have commercial potential.
There are two phases of monoclonal antibody production: the development of the hybridoma cell line and production of the monoclonal antibody from the established hybridoma cell line. Currently, the Hybridoma Core offers three methods for monoclonal antibody production from hybridoma cell lines, two in vitro methods (tissue culture and the Integra system) and one in vivo method (ascites production). The Hybridoma Core will only use the in vivo ascites method if the in vitro methods fail. The majority of hybridoma cell lines will grow in vitro; the choice of method depends on the amount, concentration, and purity of antibody required.
Polyclonal antibody production is offered on a limited basis, dependent on available space.
Peptide conjugation to KLH by a glutaraldehyde protocol.
Limiting dilution cloning of five chosen hybridoma cell lines. Collection of media samples for screening by the investigator. Minimal scale up and preparation of frozen stocks (5 vials) of clones are completed by the Core. Includes labor and supplies. This phase lasts 4-8 weeks. Freezing cells from culture expansion associated with mAb production can be frozen down for storage in Liq N2. The freezing media is 90% FBS:10% DMSO and the cells are at a concentration of 2-4 x 106 cells/ml.
We can perform antigen-capture or sandwich type ELISAs to measure antibody levels in tissue culture supernatant.
The Core will freeze back hybridomas in freezing media from actively growing cells.
Fusion of Cells
Fusion of cells to myeloma cell line. Plating of fused cells and collection of media samples for screening by the investigator. Includes labor and supplies. This phase lasts 4-6 weeks.
Integra Culture System
For production of mAb from cell lines. This system is intended for production of 30-60mg of mAb per month in the smaller system and 100-200mg of mAb in the larger system. Average concentration is 1.5mg/ml. The production schedule average is 8 weeks.
Large-Scale Antibody Production
The Core uses a static cell culture system the Integra Flask to produce high concentration (>.5mg/ml) monoclonal antibodies. This can be done in serum-free or using ultra-low IgG/IgM serum. Yields can be as high as 100mg/month/flask.
Large-Scale Antibody Purification
We can purify up to 80mg of IgG from one sample using a protein G column.
Liquid Nitrogen Storage of Cells
Storage for cloned cell lines. The stored cell lines must be mycoplasma free.
We can purify monoclonal or polyclonal antibodies using Protein G or an epitope specific affinity column.
Anti-Thymocyte Globulin (ATG) Serum or Purified ATG
Please contact the Hybridoma Core for additional services and pricing.
1 Sorvall RT7 PLUS benchtop refrigerated centrifuge for routine tissue culture work.
1 Revco -70 celsius freezer and 1 -20 celsius freezer
2 Thermo Scientific tissue culture incubators equipped with sterilization mode.
Liquid nitrogen storage
1 K Series and3 BioCane 47 Cryostorage systems capable of storing greater than 10,000 vials.
3 Nuaire Biological Safety Cabinets providing laminar flow and a sterile work environment for hybridoma tissue culture work.
Protocols & References
- BA Practical Guide to Monoclonal Antibodies. 1991. J. Eryl Liddell and A. Cryer. John Wiley & Sons, Ltd. NY, NY.
- Antibodies: A Laboratory Manual. 1989. E. Harlow and D. Lane. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
- Using Antibodies: A Laboratory Manual. 1999. E. Harlow and D. Lane. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
- Mycoplasma: Central Cell Services
- Integra Biosciences
- Peptide synthesis: Molecular Biotechnology Core
- Design for peptide antibodies: Pebio
- John Hopkins Center for Alternatives to Animal Testing
- Cleveland Clinic-approved antibody companies:
Frequently Asked Questions
How long will it take to make a monoclonal antibody?
It usually takes at least 4-6 months.
Will the monoclonal or polyclonal antibody work in all test situations?
Probably not, and this can be especially true for antibodies made against peptides. Sometimes we get lucky and get an antibody that works in most assay situations. But in many instances an antibody (or clone) works in a limited assay set, so it is important to screen with the assays for which the antibody is needed.
The Hybridoma Core provides a resource for both development and production of monoclonal antibodies, including consultation regarding all aspects of monoclonal antibody production such as epitope design and screening assays. Services are performed under strict QC guidelines appropriate for antibodies that may have commercial potential. The core uses both stationary (T flasks) and suspension (roller bottle/spinner flasks) cultures for the production of monoclonal antibodies from hybridoma cell lines. For large-scale production, hybridoma cells can also be grown in Integra flasks, which typically achieve an antibody concentration of 1mg/ml with very good antibody purity. In the production of custom monoclonal antibodies, the core routinely performs the 1st and 2nd round screens for specific antibodies using ELISA or western blot analysis.
Affinity purification of antibody from tissue culture supernatant is done frequently with purities of > 95% achieved. The HCF also offers polyclonal antibody production on a limited basis.