Rabbit Polyclonal Antibody Production Protocol (70-Day)

Standard 70-day rabbit immunization protocol for rabbit polyclonal antibody production

Abbkine has offered polyclonal antibody manufacturing services for more than four decades. Our expertise includes using a variety of different hosts for the development of polyclonal antibodies, including the most commonly used New Zealand white rabbits. Goats, sheep, rats, mouse and donkeys are also often selected.

This 2-Rabbit 70-Day protocol is optimized to produce antiserum in the shortest possible time. It is the most affordable option, popular among individual academic researchers.

The default immunization schedule uses two NZW SPF rabbits (New Zealand White rabbits that are Specific Pathogen-Free); additional rabbits are easily added to the standard order for a modest cost. Injections are subcutaneous (SQ) as emulsions in Complete Freund’s Adjuvant (CFA) or Incomplete Freund’s Adjuvant (IFA); alternative adjuvants can be used if requested. Continuation of rabbits after Day 60 is offered on a month-to-month basis. A brief protocol is listed below.

Antigen preparation, such as peptide synthesis and/or immunogen conjugation, occurs before Day 0. Injection amounts are given for a conjugated peptide antigen (e.g., KLH) or protein immunogen; MAP-peptide antigens are injected at 0.5mg throughout. Protocol Days are approximate (± 2 days).

Procedure Protocol day Description
Control serum collection Day 0 Pre-immune bleed (5 mL per rabbit)
Primary injection Day 1 Immunize with 0.50 mg of antigen in CFA, 10 SQ sites
1st booster Day 14 Boost with 0.25 mg of antigen in IFA, 4 SQ sites
2nd booster Day 28 Boost with 0.25 mg of antigen in IFA, 4 SQ sites
Serum collection Day 35 Bleed (~25 mL per rabbit)
3rd Booster Day 42 Boost with 0.25 mg of antigen in IFA, 4 SQ sites
Serum collection Day 56, 58 Two bleeds (~50 mL total per rabbit)
ELISA and shipping Day 60 ELISA titration (results available online);
Verify disposition of rabbits; decide to continue or terminate
Instruction due date Day 72 If no animal instructions are received, per diem charges will apply
Deliverables (Day 60): 12 vials crude antibody sera

  • 2 vials: 1-2 mL test aliquots pre-immune sera
  • 2 vials: 5 mL per vial pre-immune sera
  • 4 vials: 1-2 mL test aliquots post-inject sera (Day 35 and 58)
  • 4 vials: 20-50 mL per vial bulk post-inject sera (Day 35 and 58)


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