Immunization and hybridoma development schedules for custom mouse monoclonal antibody production

We offer several highly effective options for hybridoma development to produce custom monoclonal antibodies from peptide or protein antigens as part of Thermo Scientific Pierce custom antibody services.

Custom Antibody Request

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 also 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
Details and additional information

Immunization and fusion are the first two phases of the procedure for developing, producing and purifying monoclonal antibodies:

  • Phase 0: Antigen preparation
  • Phase 1: Immunization protocol and ELISA titration
  • Phase 2: Fusion protocol and screening of the positive supernatants
  • Phase 3: Subcloning
  • Phase 4 and 5: Production (cell culture) and purification

Three standard protocol options

We offer three standard protocol scales for mouse monoclonal antibody development. Additional mice are easily added to any standard order for a modest cost:

  • 5-mouse protocol: uses five Swiss Webster (SW) mice. We recommend this protocol for highly purified recombinant proteins where antibodies will be used primarily in western blotting, immunoprecipitation (IP), or ELISA. It is not recommended for peptide antigens or small proteins (<9kDa).
  • 10-mouse protocol: uses five SW mice and five BALB/c mice. We recommend this protocol for highly purified recombinant proteins and peptide-conjugates, 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 protocol: uses five SW mice, five BALB/c mice and five Black 6 mice. We recommend this protocol for 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 express protein antigens in E. coli or mammalian cells using an array of bacterial vectors or pLOC lentiviral vector. 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)

Protocol deliverables

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, 1 to 2 mL 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.

Overview of standard mouse immunization schedules and hybridoma development protocols for monoclonal antibody production. Each protocol applies to a single antigen injected into all replicate mice.

Protocol Immunization and testing Fusions and screening Delivery of hybridomas
5-mouse, 5-week 5 SW mice 2 best mice 5 best parental cell lines
10-mouse, 5-week 5 SW mice
5 BALB/c mice
3 best mice 10 best parental cell lines
15-mouse, 5-week 5 SW mice
5 BALB/c mice
5 Black 6 mice
5 best mice 15 best parental cell lines

Immunization schedule

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 4–6 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. This extension service is offered at no additional cost. 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.

Procedure Protocol day† Description
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.

Biomarker quantitation assay guide

This 72-page guide provides detailed information about different tools for protein and RNA quantitation. Download this valuable technical resource that covers technologies useful for cancer and inflammation research, immunology, neurology and more. Learn more about how antibody pairs, ELISA kits, and multiplex kits for the Invitrogen Luminex platform may help advance your research.

Download the Biomarker quantitation assay guide

Biomarker quantitation assay guide cover