Custom hybridoma and antibody production options for rat and mouse monoclonal antibodies.
We offer both rat and mouse hybridoma development options for producing custom monoclonal antibodies from synthesized peptide or recombinant protein antigens with Thermo Scientific Pierce Custom Antibody Services.
Novel immunization strategies, antigen design, and screening technologies are essential for the successful generation of monoclonal hybridoma cells lines. Our immunization protocols take advantage of using multiple strains of mice along with altered immunization routes, combinations of ligand carriers and dosing strategies to produce the most diverse monoclonal antibody population possible. This polyclonal utility is essential because diversity of clones to select for immortalization is a major limiting factor in traditional monoclonal generation. Antigens must be carefully designed, especially when dealing with peptides, to generate a robust reagent that is able to recognize conformational changes of the native protein in assay conditions.
- Monoclonal antibodies - robust protocol options for immunizing mice or rats, ELISA-titering test-bleeds, creating fusions, screening hybridomas, and delivering cell lines
- Customizable and adaptable - each protocol can be expanded or modified in a variety of ways to service specific needs for final scale, specificity or assay validation
- Deferred costs - the antibody development schedule is divided into five phases and payment stages, enabling aspects of the project scope to be changed in-process
- Full service integration - use our services to accomplish one phase of development that you do not have the resources to perform yourself, or enjoy the convenience and complete validation that accompanies our management of the entire monoclonal antibody development project:
- Antigen preparation (Phase 0)
- Immunization and hybridoma creation (Phases 1 and 2)
- Subcloning and screening to optimize cell lines (Phase 3)
- Production (cell culture) and purification (Phase 4 and 5)
- Labeling (Phase "6")
Details and Additional Information:
An entire project for monoclonal antibody development, production and purification requires approximately 4 to 6 months to complete. The process is divided in 5 phases (milestones), which are invoiced after the completion of each phase. These milestones represent exit points for you to discontinue the project if results do not meet your specific expectations. We also offer antibody labeling, which can be added as a sixth phase of the entire antibody development project. Our standard methodology is as follows:
Phase 0 – Antigen Preparation:
Protein antigen for monoclonal antibody production is expressed in E.coli or mammalian cells using a number of bacterial vectors or pLOC lentiviral vector (see additional protein expression information here). Customers may provide highly purified (>90%) protein antigens that are ready for injection. At least 4mg, 6mg and 8mg of protein antigen is required to accomplish the 5-, 10- and 15-mouse immunization and screening protocols, respectively. Alternatively, we provide services for peptide design, peptide synthesis and peptide conjugation to carrier protein for use as the immunogen.
Phase 1 – Immunization and ELISA Titration:
Animals (5, 10 or 15 mice, or 5 rats) are immunized and test-bled over a 5-week period (one primary injection and two booster injections). A titration ELISA is performed with each test-bleed. Titer and IgG-IgM composition are determined by screening with anti-IgG and anti-IgM specific secondary antibodies. ELISA results and crude antisera are sent to the customer for evaluation. See Mouse Protocols or Rat Protocols for additional details.
Phase 2 – Fusion and Screening for Positive Supernatants:
The customer chooses the best 2, 3 or 5 animals from among the original 5, 10 or 15 animals that were immunized. These best-responding animals are boosted twice more and then their spleen cells harvested for hybridoma fusion (25 to 30 plates per fusion). An ELISA assay is used to screen all positive supernatants, 1 to 2mL of which are also sent to the customer for evaluation. Cell lines are preserved (expanded and frozen) and the best parental cell lines are delivered to the customer as frozen stocks. See Monoclonal Antibody ELISA Testing for additional details.
Phase 3 – Subcloning before Production:
Positive parental cell lines can be subcloned through limiting dilution cloning to obtain daughter cell lines. Subcloning is important for the long-term stability of clones and to ensure that cells are truly monoclonal and maintain clonality over their production life. Up to five daughter cell lines are provided after subcloning. Daughter cell lines may all react identically. Cell viability is also confirmed through freeze-thaw.
Two subcloning options are offered. Single-cycle cloning involves making a single round of limiting-dilution subcloning to pick out consistent producers with confirmed isotype and production. Multi-cycle complete clonality cloning involves performing multiple rounds of limiting-dilution subcloning until all wells are growing at the same rate, excreting antibody at the same concentration and have the same confirmed isotype.
Phase 4 and 5 – Production and Purification:
Once high-quality, antibody-producing clones have been developed, monoclonal antibodies can be produced from them by either (1) cell culture in a roller bottle to yield antibody-containing supernatant, or (2) mouse abdominal culture to yield antibody-containing ascites fluid. Each liter of cell culture yields approximately 10 to 30mg of antibody. Each mouse culture yields 2 to 10mg in 2 to 3mL of neat ascites fluid.
Several options are available for monoclonal antibody purification from supernatant or ascites. Most customers prefer simple affinity purification with Protein A/G resin, but we also can purify using anti-immunoglobulin antibodies or ion-exchange columns. See Monoclonal Antibody Production and Purification Options for additional details.