The following is a general outline of procedures used by vivarium staff for the production of polyclonal antibodies in rabbits. The technical services are done on a recharge basis and are in addition to the standard per diem. A work order request must be completed by the investigative staff for all procedures, the forms are available at the vivarium office. The fee schedule for antibody production follows.

The PI must have a CARC approved research/teaching protocol application before any animals can be procured and before any work can be done. A boilerplate protocol that contains the procedures used by the vivarium is available from the CARC web site.

Each immunogen is different and no single immunization procedure will be optimal for every antigen. Rabbits are genetically diverse and each responds uniquely to the procedures. Antibody isotype predominance and individual epitope responses can be affected by immunization procedures. The procedures described here generate good results in the majority of cases, based on reports in the published literature. Further assistance is available from the campus veterinarian.

General Procedures Followed by the Vivarium.

1. Rabbits should be allowed to acclimate to the new surroundings for one full week. Immunization procedures can begin at that time.
2. The investigator provides the vivarium staff with 50:50 emulsion of antigen and adjuvant. Most protocols use 1.0 ml emulsion of Freund's complete adjuvant (FCA) for the initial immunization; however, other adjuvants should be considered, especially for large and complex antigens that require lesser adjuvant activity. The antigen should be Millipore filtered for sterility prior to mixing with adjuvant and must not contain any polyacrilimide gel when FCA is used.
3. A 10 ml pre-immunization blood sample is taken and the initial immunization is given. These generally occur on the same day. The procedures are generally done under 1 mg/kg acepromazine for sedation. A maximum of 1.0 ml of emulsion is injected subcutaneously in 0.1 ml injections.
4. The first booster immunization is given at three to four weeks for Freund's adjuvant. Freund's complete adjuvant must not be used as adjuvant following the initial injections. All booster immunizations must be Freund's incomplete adjuvant. The volumes and techniques used are the same as for the initial immunization.
5. Two to three weeks later a test bleed of 10 mls is recommended. Serum from that bleed should be titered to assess the need for further booster injections.
6. If antibody levels are not adequate a second booster immunization can be done. All subsequent booster immunizations should be based on the adequacy of measured serum antibody titers. Once a usable antibody titer is achieved, booster immunizations are required on an infrequent schedule.
7. Blood samples for serum isolation are generally collected about 2 weeks after an immunization and are generally a volume with a weight equivalent to 1% of body weight (30 mls for a typical 3 kg rabbit). Blood is collected in tubes without anticoagulant and hence a clot with serum is recovered. The lab is called when the tubes are ready for pick-up.

When large quantities of antibody are needed or there is no longer a need for an on-going antibody supply, a maximal blood sample can be collected as the rabbit is euthanized. This procedure involves general anesthesia (35-40 mg ketamine and 5 mg Xylazine per kg body weight; or Isoflurane gas anesthesia) and terminal blood collection by cardiac puncture.

The vivarium can assist with developing other immunization procedures and can provide the technical services required for other techniques.

Effective 8/1/98

I. Availability and scheduling

Veterinary Technicians are available to assist researchers with production of antibodies prescribed by the clinical veterinarian. Antibody production must be scheduled with the veterinarian.

II. Fee Schedule

• The researcher must purchase the rabbit and pay for the per diem for its care.
• The researcher must purchase the adjuvant.
• The researcher is responsible for the mixing of the adjuvant and the antigen.
• The vivarium staff will administer immunizations and perform phlebotomies under the CARC approved standard operating procedure for the production of polyclonal antibodies from rabbits.
• All charges for injections and phlebotomies will be based upon a minimum billing unit of 1/4 hour @ $4.00. The hourly rate will be $16.00.

1. Pre-immunization phlebotomy. Up to 1% of the body weight in blood volume per week allowable (30 mls for a typical 3 kg rabbit). Multiple weeks of pre-immunization phlebotomy possible.
2. Immunization injection. Researcher responsible for the mixing of the antigen and adjuvant.
3. Must use CARC approved protocol for antibody production. Other protocols are possible but will require researcher to justify change in protocol and researcher must submit a complete CARC application, i.e., researcher cannot use the "boilerplate" application. CARC approved protocol appended to the end of this document.
4. Phlebotomy at prescribed intervals to check titer.
5. Re-immunization at prescribed intervals necessary to achieve sufficient titer.
6. Phlebotomy at prescribed intervals to collect serum. Blood is collected in tubes without anticoagulant and hence a clot with serum is recovered. The researcher's lab is called when the tubes are ready for pick-up. Up to 1% of the body weight in blood volume per week allowable (30 mls for a typical 3 kg rabbit). After eight weeks or upon significant decrease in hematocrit, the veterinarian may elect to wait 30 days before the next phlebotomy.
7. When large quantities of antibody are needed or there is no longer a need for an on-going antibody supply, a maximal blood sample can be collected as the rabbit is euthanized. This procedure involves general anesthesia and terminal blood collection by cardiac puncture.

A minimum of 15 minutes will be charged for each procedure, and billing units are 1/4 hour for anything over 15 minutes. A minimum of 1/2 hour ($8.00) will be assessed for any phlebotomy and a minimum of 1 hour ($16.00) for terminal blood collection.

Estimate of Costs

You may figure the cost of producing an antibody by adding the total fees charged. Use

the cost-estimation sheet below to figure your cost. An example for a three-month period

for two rabbits is given below.

Number of Rabbits: 2

Number of days: 100

Initial fee: 2 animals x $35.00 each = $ 70.00

Pre-bleeds (optional): 2 animals x $8.00 each = $ 16.00

Cost of Fruend's complete/incomplete adjuvant @ approx. $3.00 per injection = $24.00

Immunizations: 2 animals x 4 immunizations per animal x $8.00 each = $64.00

Test Bleedings: 2 animals x 4 test bleeding per animal x $8.00 each = $ 64.00

Bleedings: 2 animals x 3 bleedings per animal x $8.00 each = $48.00

Per Diem: 2 animals x 100 days x $1.40 per day = $ 280.00

Terminal bleed: 2 animals x one hour time = $32.00

Total Cost $ 598.00

Note: The actual cost may be more or less than the example if adequate titers are achieved quickly or if one rabbit is used, if more bleedings are done, or if the schedule should extend beyond 100 days.

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