Immunization and hybridoma development schedules for custom mouse monoclonal antibody production
Mouse immunization and spleen fusion to create hybridoma cell lines are two phases of an entire procedure for developing, producing, and purifying monoclonal antibodies. We offer 5-mouse (1-strain), 10-mouse (2-strain), and 15-mouse (3-strain) protocols for immunization and hybridoma development. We perform and report in-process ELISA testing and send samples to customers for evaluation, allowing customers to select which animals to use for fusion and which cell lines to subclone, expand, and deliver.
- Mouse monoclonal antibodies—optimized and efficient protocols for immunizing mice, creating fusions, screening hybridomas, and delivering cell lines
- Guaranteed titer—after completing the initial immunization schedule, we will continue to boost mice at no additional charge until a quantifiable minimum titer is achieved
- Customizable—you decide (based on our screening results and your own testing) which immunized mice to fuse and which cell lines to subclone and expand
- Full-service integration—combine these protocols with our complete set of services for initial antigen preparation and subsequent antibody production and purification
An entire project for monoclonal antibody development, production, and purification requires approximately 6 months to complete. The process is divided into 5 phases (milestones), which are invoiced after the completion of each phase. These milestones represent exit points for discontinuing the project if results do not meet your specific expectations. Antibody labeling can be added as a sixth phase to the antibody development project.
Phase 1: Antigen preparation
Customers may provide highly purified (>90%) protein or peptide antigens that are ready for injection. Alternatively, we provide services for protein production, peptide design, peptide synthesis, and peptide conjugation to carrier protein for use as the immunogen.
Phase 2: Immunization and ELISA titration
Animals are immunized and test-bled over a 6-week period (one primary injection and two booster injections). A titration ELISA is performed with each test-bleed. ELISA results and crude antisera are sent to the customer for evaluation.
Phase 3: Fusion and screening for positive supernatants
The best-responding animals are boosted twice more; then their spleen cells are harvested for hybridoma fusion (10 plates per fusion). An ELISA assay is used to screen the positive supernatants, 1 to 2 mL of which are sent to the customer for evaluation. Cell lines are preserved (expanded and frozen) and stored during customer evaluation.
Phase 4: Subcloning before production
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. Multi-cycle complete clonality cloning involves performing multiple rounds of limiting-dilution subcloning until all wells are growing at the same rate, are secreting antibody at the same concentration, and have the same confirmed isotype.
Phase 5: Production and purification
Monoclonal antibodies are produced in cell culture in roller bottles to yield antibody-containing supernatants. Each liter of cell culture yields approximately 10 to 30 mg of antibody. Low-endotoxin and serum-free culturing are available on request, as well as high-volume production.
Depending on the isotype of the monoclonal antibody, affinity purification is performed using Protein A or Protein G resin.
Phase 6: Labeling
We offer a wide variety of custom fluorescent antibody and protein labeling options.
Three standard protocol options
|Protocol||Immunization and testing||Fusions and screening||Delivery of hybridomas||Antigen recommendation|
|5-mouse, 5-week||5 SW mice||2 best mice||5 best parental cell lines||Purified proteins where antibodies will be used primarily in western blotting, immunoprecipitation (IP), or ELISA. It is not recommended for peptide antigens or small proteins (<9 kDa).|
|10-mouse, 5-week||5 SW mice|
5 BALB/c mice
|3 best mice||10 best parental cell lines||Purified proteins and small peptides where antibodies will be used primarily in western blotting (WB) or immunohistochemistry (IHC), but are also needed for multiple applications where epitope structure is extremely important (IP, capture, etc.).|
|15-mouse, 5-week||5 SW mice|
5 BALB/c mice
5 Black 6 mice
|5 best mice||15 best parental cell lines||Peptide-conjugates, compounds, purified proteins and small antigens where antibodies will be used primarily in a quantitative format where epitope structure is extremely important (IP, capture, ELISA, multiplex, etc.).|
Required antigen preparation
Purified antigen must be prepared (Phase 0) in advance of the immunization and fusion protocols. We offer antigen design and synthesis services. For monoclonal antibody development, we can design a suitable small peptide antigen, or we can express protein antigens in E. coli. Alternatively, customers may provide highly purified (>90%) protein antigens that are ready for injection. The amounts of antigen that must be prepared in advance of each protocol are as follows:
- 5-mouse protocol: 4 mg total (2 mg to immunize and boost / 2 mg to screen)
- 10-mouse protocol: 8 mg total (4 mg to immunize and boost / 4 mg to screen)
- 15-mouse protocol: 12 mg total (6 mg to immunize and boost / 6 mg to screen)
The outputs and deliverables associated with the three protocols are summarized in the table below. Mice (5, 10, or 15 animals) 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, and ELISA results and crude antisera are sent to the customer for evaluation.
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 (10 plates per fusion). An ELISA assay is used to screen all positive supernatants with 1 mL to be sent to the customer for evaluation. Cell lines are preserved (expanded and frozen). The customer chooses the best parental cell line(s) for subcloning. An ELISA assay is used to screen the subcloned cell lines which are the final monoclonal cell lines. Samples of 1 mL supernatant will be sent to the customer for evaluation. Finally, once the customer chooses which cell lines will go to production, the process will be completed, and final purified monoclonal antibody and cell line will be delivered.
All three mouse protocols use the same immunization schedule and method. Primary and first booster injections are IP as emulsions in Freund's Complete Adjuvant (CFA) or Incomplete Freund's Adjuvant (IFA); alternative adjuvants can be used if requested. Final boosts before fusion are intraperitoneal (IP) and intravenous (IV). Total development time is approximately 6 months. Depending on the initial ELISA titration results, the mice may need additional boosts and bleeds in order to generate titers that meet our minimum requirement for fusions. Additional subcloning and monoclonal production options are available for an additional charge; decisions about these options can be made at any time during the project, based on in-process titer and screening results.
|Control serum collection||Day 0||Pre-immune bleed (0.2–0.5 mL per mouse)|
|Primary injection||Day 1||Immunize with 0.1 mg antigen in CFA, IP|
|Booster injections||Days 14, 28||Boost with 0.1 mg antigen in IFA, IP|
|Test bleeds||Day 42||Test-bleed (0.2–0.5 mL per mouse)|
|ELISA titration||Day 43–60||ELISA titration of pre-immune and test-bleeds;|
Data delivery, customer evaluation and mouse selection
|Pre-fusion booster||Day 62||Boost with 0.1 mg antigen in saline, IP|
|Pre-fusion booster||Day 64||Boost with 0.1 mg antigen in saline, IV|
|Fusion||Day 66||Fuse myeloma cells and spleen cells|
|ELISA and subcloning||Day 80||Screen clones, then subclone to ensure monoclonal lines|
|ELISA screening||Day 94||Screen clones, then freeze stocks;|
Send supernatants to customer for evaluation
|Expansion and delivery||Day 100||Expansion and freeze-down of chosen parental stocks|
|†After Day 35, the indicated days in this schedule are approximate because they depend upon customer evaluation to select animals and samples for continuation or termination.|