STANDARD OPERATING PROCEDURE (SOP) FOR POLYCLONAL ANTIBODY PRODUCTION IN RABBITS

Standard Operating Procedure (SOP) for Polyclonal Antibody Production in Rabbits

PURPOSE

This SOP describes the procedure for producing polyclonal antibodies. The production of polyclonal antibodies is provided as a fee-for-service through the Comparative Medicine and Animal Resources Centre (CMARC).

RESPONSIBILITY

- Principal Investigator (PI)

- Veterinary Care Staff

MATERIALS

1. Freund's Complete Adjuvant (FCA)

2. Freund's Incomplete Adjuvant (FIA)

3. Syringes and needles

4. Microchip identification system

5. Medical records

6. Acepromazine

7. Butorphanol

8. EMLA cream

9. Blood collection materials

10. Chlorhexidine

11. Ketamine

12. Xylazine

13. Glycopyrrolate

PREPARATION OF IMMUNOGEN

- The PI must prepare an immunogen that is:

1. Non-toxic

2. Sterile

3. pH within physiological limits

4. Free of pyrogens

5. Easily passed through a 20G needle

**Note**: Proteins in polyacrylamide gel may cause adverse reactions at the injection site. Use another method of purification or dilution when possible.

- The PI must provide 4 samples of the immunogen (labeled 1-4), each consisting of 200-500 micrograms of antigen in sterile PBS in a volume of 500 µl per rabbit.

PROCEDURES

Adjuvant

1. Use an adjuvant to increase the immunological response to poor antigens.

- **Note**: When used with a strong antigen, the adjuvant may induce an overt local inflammatory response.

2. Use Freund's Incomplete Adjuvant (FIA) and Freund's Complete Adjuvant (FCA). In case of overt reaction, other adjuvants are available for use.

3. Administer FCA only once for the primary injection. Do not repeat. Use only FCA with a concentration of 0.5 mg/ml of mycobacteria or less.

4. Use FIA for all secondary immunizations.

5. Combine the antigen and the adjuvant using two syringes and a locking connector (e.g., 3-way stopcock) and emulsify until it no longer separates.

RECORDKEEPING

- Record the following information in the animal's medical record:

1. Name of immunogen

2. Adjuvant used

3. Route of administration

4. Site of injection

5. Volume of injection

6. Date of injection

- Record blood collection volume and site, as well as body weight, general condition, and appearance of the injection site.

ANIMAL SELECTION

- Use young, adult female-specific pathogen-free rabbits.

WEEKLY MAINTENANCE

1. Remove bedding and disinfect pens and surrounding walls.

2. Change food hoppers/bowls and litter pans once every two weeks. Clean and replace environmental enrichment devices at the same time.

3. Change the cage bank every two weeks at a minimum. Only transfer one animal at a time to avoid unintentional mix-ups.

4. Mop floors with disinfectant solution.

5. Flush the automatic watering system once per week.

6. Check light timers.

7. Flush the floor drains.

MONTHLY MAINTENANCE

1. Change the automatic water system pressure-reducing station filter.

2. Wipe down all surfaces, such as sinks, door jams, doorknobs, cart tops, and shelves with disinfectant solution.

3. Clean room exhaust grills.

BI-ANNUAL MAINTENANCE

1. Wash all walls with disinfectant solution.

SPECIAL PROCEDURES

Sick or Injured Animals

1. Place a veterinary care card on the cage or pen of the sick or injured animal.

2. Complete an illness/injury report and submit it to the veterinary care staff.

PREPARATION OF DISINFECTANT SOLUTION

1. Wear personal protective equipment when preparing and using disinfectant solutions.

2. Dilute according to the manufacturer's recommendations.

3. Clean surfaces prior to disinfecting to remove any organic material.

4. Apply disinfectant and allow for adequate contact time.

END OF THE DOCUMENT

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