Immunization and hybridoma development schedule for custom rat monoclonal antibody production

We offer a customizable option for rat hybridoma development (rat immunization and hybridoma fusion) to produce custom monoclonal antibodies from peptide or protein antigens as part of Thermo Scientific Pierce custom antibody services.

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Rat immunization and fusion to create hybridoma cell lines are two phases of an entire procedure for developing, producing and purifying rat monoclonal antibodies. We offer an effective 5–rat protocol 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.


  • Rat monoclonal antibodies—optimized and efficient protocol for immunizing rats, creating fusions, screening hybridomas, and delivering cell lines
  • Guaranteed titer—after completing the initial immunization schedule, we will continue to boost rats 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 rats to fuse and which cell lines to subclone and expand
  • Full service integration—combine the rat protocol with our complete set of services for initial antigen preparation and subsequent antibody production and purification
Details and additional information

Immunization and fusion comprise the first two phases of protocol 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

Rat monoclonal protocol

Our standard protocol for rat monoclonal antibody development uses five Sprague Dawley rats, but additional rats are easily added to any standard order for a modest cost. The outputs and deliverables associated with the protocol are summarized in the table below.

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, and ELISA results and crude antisera are sent to the customer for evaluation.

The customer chooses the best two rats from among the original five 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 2 mL of which are also sent to the customer for evaluation. Cell lines are preserved (expanded and frozen) and the five best parental cell lines are delivered to the customer as frozen stocks.

Rat immunization and hybridoma development protocol for rat monoclonal antibody production

Protocol Immunization and testing Fusions and screening Delivery of hybridomas
5-rat, 11-week 5 Sprague Dawley rats 2 best rats 5 best parental cell lines

Required antigen preparation

Purified antigen must be prepared (Phase 0) in advance of the immunization and fusion protocol. We offer antigen design and synthesis services. For monoclonal antibody development, we express protein antigens in E.coli or mammalian cells using a number of bacterial expression vectors quartet or pLOC lentiviral vector. Alternatively, customers may provide highly purified (>90%) protein antigens that are ready for injection. The 5-Rat Protocol requires preparation of 4mg of purified protein antigen (2mg for immunizations, 2mg for screening).

Immunization schedule

Primary and first booster injections are (IF) 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 rats 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 anytime during the project based on in-process titer and screening results.

Immunization and fusion schedule for rat monoclonal antibody development.</b>&nbsp;Antigen preparation, such as protein expression, occurs before Day 0. Injection amounts are given for a conjugated peptide antigen (e.g., KLH), MAP-peptide, or protein immunogen.

Procedure Protocol day† Description
Control serum collection Day 0 Pre-immune bleed (0.2-0.5 mL per rat)
Primary injection Day 1 Immunize with 0.1 mg antigen in CFA, SQ 4 sites
Booster injections Days 14, 21 Boost with 50 µg antigen in IFA, SQ 4 sites
Test bleeds Day 35 Test bleed (0.2-0.5 mL per rat)
ELISA titration Day 36-60 ELISA titration of pre-immune and test bleeds;
Data delivery, customer evaluation and rat 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.
Protein quantitation immunoassays