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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.

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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—easily add additional rabbits to the default protocol; track and manage projects online with OpenProject Tool: you decide which animals to continue or terminate (see Extensions below)
  • 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)

2-rabbit 70-day 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); 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 (see Protocol Extensions below).

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

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)

2-rabbit 90-day 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); 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 77 is offered on a month-to-month basis (see Protocol Extensions below).

Standard 90-day rabbit immunization protocol for rabbit polyclonal antibody production

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 0.25 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)

2-rabbit 120-day 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 a 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); 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 106 is offered on a month-to-month basis (see Protocol Extensions below).

Standard 2-rabbit 120-day immunization protocol for rabbit polyclonal antibody production

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 (1.0mg for 4th Booster). 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)

Protocol extensions

Several pre-defined (yet customizable) options are available for extending or continuing the rabbit immunization and serum-collection schedule. The purchase decision to continue antibody production can be made at any time before scheduled termination. For an additional cost, exsanguination bleeds may be obtained at the time of termination, yielding approximately 50mL of additional serum per animal.

One-month extension protocol for rabbit antibody production.

Procedure Protocol Day Description
1st Booster Day 1 Boost with 0.10mg antigen in IFA, 4 SQ sites
Serum Collection Day 14 Bleed (~25mL per rabbit)

Deliverables (per rabbit): 2 vials crude antibody sera

  • 1 vial: 1-2mL test aliquots post-inject sera
  • 1 vial: 25mL per vial bulk post-inject sera

High dose, one-month extension protocol for rabbit antibody production

Procedure Protocol day Description
1st Booster Day 1 Boost with 0.20 or 0.50 mg antigen in IFA, 4 SQ sites
Serum Collection Day 14 Bleed (~25 mL per rabbit)

Deliverables (per rabbit): 2 vials crude antibody sera

  • 1 vial: 1-2 mL test aliquots post-inject sera
  • 1 vial: 25 mL per vial bulk post-inject sera

Aggressive, one-month extension protocol for rabbit antibody production

Procedure Protocol day Description
1st booster Day 1 Boost with 0.50 mg antigen in IFA, 4 SQ sites
2nd booster Day 7 Boost with 0.20 mg antigen in IFA, 4 SQ sites
Serum collection Day 21 Bleed (~25 mL per rabbit)

Deliverables (per rabbit): 2 vials crude antibody sera

  • 1 vial: 1-2 mL test aliquots post-inject sera
  • 1 vial: 25 mL per vial bulk post-inject sera

One-month auto-termination extension protocol for rabbit antibody production

Procedure Protocol day Description
1st booster Day 1 Boost with 0.10 mg antigen in IFA, 4 SQ sites
Serum collection Day 14 Bleed (~25 mL per rabbit)
Serum collection Day 16 Bleed (~25 mL per rabbit)

Deliverables (per rabbit): 2 vials crude antibody sera

  • 1 vial: 1-2 mL test aliquots post-inject sera
  • 1 vial: 50 mL per vial bulk post-inject sera

Termination bleed protocol for rabbit antibody production

Procedure Protocol day Description
Serum collection Day 1 Bleed (~25 mL per rabbit)
Serum collection
and termination
Day 3 Bleed (~25 mL per rabbit);
Animal euthanized at our discretion

Deliverables (per rabbit): 2 vials crude antibody sera

  • 1 vial: 1-2 mL test aliquots post-inject sera
  • 1 vial: 50 mL per vial bulk post-inject sera

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.

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