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Immunization and serum-collection procedures for custom rabbit polyclonal antibody production

We offer three standard protocol packages and several extension options (immunization and serum collection schedules) for rabbit polyclonal antibody production within our complete array of Thermo Scientific Pierce custom antibody services.

Custom Antibody Request

Highlights

  • Custom rabbit antibodies—three standard protocol options for immunizing rabbits, collecting serum, testing bleeds, reporting titer results and delivering antisera
  • Rabbit titer guarantee—if you allow us to oversee design and production of the peptide and/or protein antigen used for injection, we guarantee to reach a minimum rabbit antiserum titer of 1:50,000 as measured with our ELISA assay
  • Track and customize project—track and manage projects online with the OpenProject Tool: you decide which animals to continue or terminate
  • Full service integration—combine this immunization service with our complete set of services for antigen preparation and antibody purification (see Custom Polyclonal Antibodies pages for details)

70-day rabbit immunization protocol

Of the three standard options for rabbit polyclonal antibody production, this 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). 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.

Antigen preparation, such as peptide synthesis and/or immunogen conjugation, occurs before Day 0. 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)

90-day rabbit immunization protocol

Of the three standard options for rabbit polyclonal antibody production, this protocol is optimized to maximize speed, titer, and volume for a competitive price. Originally developed and adopted by the NIH, this procedure meets the minimum requirements mandated by most pharmaceutical companies.

The default immunization schedule uses two NZW SPF rabbits (New Zealand White rabbits that are Specific Pathogen-Free). 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.

Antigen preparation, such as peptide synthesis and/or immunogen conjugation, occurs before day 0. 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 0.25 mg antigen in CFA, SQ 4 sites
1st booster Day 14 Boost with 0.10 mg antigen in IFA, 4 SQ sites
Serum collection Day 28 Bleed (~25 mL per rabbit)
2nd booster Day 42 Boost with 0.10 mg antigen in IFA, 4 SQ sites
Serum collection Day 56 Bleed (~25 mL per rabbit)
3rd booster Day 56 Boost with 0.10 mg antigen in IFA, 4 SQ sites
Serum collection Day 70, 72 Two bleeds (~50 mL total per rabbit)
ELISA and shipping Day 77 ELISA titration (results available online);
Verify disposition of rabbits; decide to continue or terminate
Instruction due date Day 90 If no animal instructions are received, per diem charges will apply

Deliverables (Day 77): 16 vials crude antibody sera

  • 2 vials: 1–2 mL test aliquots pre-immune sera
  • 2 vials: 5 mL per vial pre-immune sera
  • 6 vials: 1–2 mL test aliquots post-inject sera (Day 28, 56, and 72)
  • 6 vials: 20–50 mL per vial bulk post-inject sera (Day 28, 56, and 72)

120-day rabbit immunization protocol

Of the three standard options for rabbit polyclonal antibody production, this protocol is optimized to maximize titer and volume by ensuring that sufficient time is provided for full maturation of the secondary immune response and serum antibody production in each rabbit. Based on the method originally developed by Baines and Korn (1990), this procedure exceeds the minimum requirements mandated by most pharmaceutical companies.

The default immunization schedule uses two NZW SPF rabbits (New Zealand White rabbits that are Specific Pathogen-Free). 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.

Antigen preparation, such as peptide synthesis and/or immunogen conjugation, occurs before Day 0. 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 0.25 mg antigen in CFA, SQ 4 sites
1st booster Day 14 Boost with 0.10 mg antigen in IFA, 4 SQ sites
2nd booster Day 28 Boost with 0.10 mg antigen in IFA, 4 SQ sites
Serum collection Day 42 Bleed (~25 mL per rabbit)
3rd booster Day 56 Boost with 0.10 mg antigen in IFA, 4 SQ sites
Serum collection, ELISA titer Day 70 Bleed (~25 mL per rabbit)
4th booster Day 84 Boost with 0.10 mg antigen in IFA, 4 SQ sites
Serum collection, ELISA titer Day 98, 100 Two bleeds (~50 mL total per rabbit)
ELISA and shipping Day 106 ELISA titration (results available online);
Verify disposition of rabbits; decide to continue or terminate
Instruction due date Day 120 If no animal instructions are received, per diem charges will apply

Deliverables (Day 106): 16 vials crude antibody sera

  • 2 vials: 1–2 mL test aliquots pre-immune sera
  • 2 vials: 5 mL per vial pre-immune sera
  • 6 vials: 1–2 mL test aliquots post-inject sera (Day 42, 70, and 100)
  • 6 vials: 20–50 mL per vial bulk post-inject sera (Day 42, 70, and 100)

Rabbit titer guarantee

Custom rabbit antibodies manufactured by the Seller are guaranteed to reach a minimum titer of 1:50,000 by indirect ELISA assay for at least one rabbit so long as the following conditions are met: (a) Seller designed and produced the peptides and/or proteins used for immunization; (b) the design criteria did not contain Buyer mandated limitations, including but not limited to, cross reactivity requirements, limited protein sequence region, and/or post-translationally modified or cleaved peptides; and (c) the antibodies were generated in rabbits. If the antibody does not reach the minimum titer and all the above criteria are met, Seller will either (y) boost one animal (minimal) at no charge or (z) alter the carrier or antigen to boost one animal (minimal) for up to two months free of charge. Once one animal reaches the minimum titer of 1:50,000 the guarantee will be met. If a 1:50,000 titer cannot be achieved, the Buyer will be provided one additional custom antibody to be ordered free of charge with a re-design of the peptide and/or protein used for immunization. All ancillary services requested by the Buyer on the new project such as purification and extensions will be charged a la carte at the discretion of the Seller.

Maintenance fees

Upon completion of any antibody protocol, Buyers are provided seven business days to evaluate the antibody quality and provide instructions on the requested animal disposition. If no instructions are received from the Buyer, animals will be placed on hold and begin incurring daily maintenance fees according to then current fee schedule. If more than 30 days pass without receipt of instructions, animals will be scheduled for termination. An invoice for accrued charges will be sent to Buyer for all fees incurred. Payment of such fees is required prior to provision of additional services.