CMP Antibody Production Service

Transcription

1 CMP Antibody Production Service Polyclonal antibody production has become an essential part of many research projects at Texas A&M. Standardization of production procedures and the performance of the procedures by skilled personnel that perform such tasks on a daily basis result in better quality care and more attention to the welfare of production animals. CMP will offer this service to TAMU investigators to: 1) Provide a centralized service that allows the use of standardized procedures, materials and methods to optimize polyclonal antibody production in individual animals. 2) Optimize conditions required to house and maintain antibody production animals. 3) Allow maintenance of animals under high quality handling, use and care conditions. 4) Use fewer animals for antibody production. Investigators wishing to use CMP Antibody Production Program should contact Ms. Andrea Taylor at ; or abtaylor@vprmail.tamu.edu You should receive a copy of the Antigen Submission Request form as well as a schedule of costs. The completed Antigen Submission form should be returned to Ms. Taylor - Mailstop 4473; Fax: The form will be reviewed by one of the CMP veterinary staff or Dr. Surya Waghela of the Antibody Core Laboratory, College of Veterinary Medicine and any questions concerning the antigens to be used will be addressed with the investigator submitting the request. For recombinant antigens Appropriate RDNA forms for recombinant Antigen production must have been approved as required by the RDNA committee. A copy of the approved RDNA form should be provided to the CMP staff for their records if direction and safety concerns are required when handling the antigen. Animals will be purchased from CMP approved vendors, and allowed to overcome shipping stressors and adjust to their new environment for 5-7 days. Animals will be inoculated by CMP staff with an antigen preparation supplied by investigators. Antigens supplied to CMP for inoculation must be aseptically prepared or sterilized by filtering. Specific volumes and concentrations are addressed under each species. Coordination of antigen delivery, animal work, blood collections and harvests will be handled by Ms. Taylor (Andrea). Contact information: Phone: (CMP Administrative offices) Fax: ; or abtaylor@vprmail.tamu.edu Immunization protocols: The standard immunization protocol with all species will use Titermax (or Titermax Gold )as the adjuvant. If the animal does not respond as expected, or if the Titermax protocol is known to be a poor choice for the specific antigen, an alternative adjuvant selection will be made following consultation with the investigator, an immunologist (Dr. Waghela) in the Antibody Core Laboratory, College of Veterinary Medicine and/or CMP veterinary staff (Dr. Browder). Alternative adjuvant schedules are listed on charts below the respective species on the following pages. Some projects may require the use of Complete and Incomplete Freund s adjuvant. This adjuvant combination has been implicated in potentially causing painful lesions in animals during antibody production. Each antigen will be evaluated on a case by case basis and the most appropriate application for antibody response will be used. Whenever possible, other adjuvant combinations (Ribi, Titermax, Aluminum Hydroxide, Saponin, others) will be used prior to the use of Freund s adjuvant. However, extremely small proteins, autologous proteins, or other antigens identified by the Antibody Production Core Laboratory (Dr. Waghela), that historically do not respond well may require Freund s as a first option. In these cases, Freund s will be used in schedules that will reduce tissue inflammatory reactions and minimize discomfort to the animal. Some animals that respond well to an antigen prep, yet remain healthy (active animals in good physical condition with normal food and water intake and normal eliminations) and do not exhibit ill effects relating to the procedures may be kept for extended periods of time (1-5 years). These animals would receive periodic boosts (1-12 times per year) to maintain production levels with harvest collections as needed by the investigative group (but within bounds previously established in this AUP).

2 RABBITS: mg of the antigen/antigen buffer in up to 0.5 ml volume should be supplied to CMP. A maximum total volume of adjuvant + antigen-buffer mix = 1.0 ml. Please note: it is helpful if a small amount of extra antigen/antigen buffer can be supplied to CMP to account for volume loss in syringe and needle hubs. Primary Inoculation: TiterMax & TiterMax Gold /Antigen-buffer (1:1 v/v) - Total volume 1.0 ml,, 0.2 ml/site at 5 sites. Booster Inoculation: 3-6 weeks intervals with antigen in buffer or in the same adjuvant, IM, 0.4 ml/site at 2 sites until sufficient antibody levels have been reached. Usually this occurs after the third boost. Subsequent boosts may be given periodically to maintain the antibody levels for regular bleeding. The need for such boosts will be dictated by antibody levels measured in test samples. Pre-bleeds (one time, 5 cc) or Test bleeds (approximately 3cc) from the central ear artery, marginal ear vein or jugular vein every 2-3 weeks. Harvest bleeds from the central ear artery, marginal ear vein or jugular vein will be done 1-3 times every 30 days (once every two weeks), with the total 30 day volume not to exceed 1% of the body weight in volume(approximately 15% of the Total Blood volume). If additional collections are necessary, the animal will receive fluid replacement and their packed Red blood cell volume will be monitored under the guidance of CMP veterinary staff to ensure the health and well-being of the animal. Terminal bleeds will be performed with the animal under anesthesia (Ketamine mg/kg IM + Xylazine 2-5 mg/kg IM) followed by euthanasia with a barbiturate overdose. Product supplied to the investigator will be clotted blood. Centrifugation and collection of serum from the clotted blood will be the responsibility of the investigator. ALTERNATIVE ADJUVANT USE in RABBITS: Complete Freund s Adjuvant**- Incomplete Freund s Adjuvant- RIBI - Total volume 1.0 ml Alhydrogel -Antigen/buffer (10:1 to 1:1 v/v) ID ml/site max. 6 sites 1 st inoculation only ml/site max. 6 sites Booster inoculations 0.25 ml/site saline or in IFA, given /IM (see next) at an interval of 3-4 weeks with the antigen in the same adjuvant given with the antigen in saline or in the same adjuvant given IM (0.25 ml/site at ) Saponin (3 mg/ml) in Antigen-buffer mix with antigen in buffer or in the same adjuvant given IM (0.25 ml/site at ) *ID=Intradermal; =Subcutaneous; IM=Intramuscular

3 SHEEP & GOATS: mg of the antigen in up to 2.0 ml. A maximum total volume of adjuvant + antigen buffer mix = 4.0 ml. Primary Inoculation: TiterMax & TiterMax Gold /Antigen-buffer (1:1 v/v) - Total volume 4.0 ml, IM, ml/site at. Booster Inoculations: 3-6 weeks intervals with antigen in buffer or in the same adjuvant given IM, 0.8 ml/site at until sufficient antibody levels have been reached. Usually this occurs after the third boost. Subsequent boosts may be given periodically to maintain the antibody levels for regular bleeding. The need for such boosts will be dictated by antibody levels measured in test samples. Prebleeds and Test bleeds (5-10cc) from the jugular vein every 2-3 weeks. Harvest bleeds from the jugular vein will be done 1-3 times every 30 days, with the total 30 day volume not to exceed 1% of the body weight in volume (approximately 15% of the Total Blood volume). If additional collections are necessary, the animal will receive fluid replacement and their packed Red blood cell volume will be monitored under the guidance of CMP veterinary staff to ensure the health and well-being of the animal. Product supplied to the investigator will be clotted blood. Centrifugation and collection of serum from the clotted blood will be the responsibility of the investigator. ALTERNATIVE ADJUVANT USE in SHEEP and GOATS: Complete Freund s Adjuvant**- Incomplete Freund s Adjuvant- RIBI - Total volume 1.0 ml IM IM ml/site max. 1 st inoculation only max. Booster inoculation 0.25 ml/site saline or in IFA, given IM (see next) at an interval of 3-4 weeks with the antigen in the same adjuvant given Alhydrogel -Antigen/buffer (10:1 to 1:1 v/v) 1.0 ml/site with the antigen in saline or in the same adjuvant given IM at Saponin (3 mg/ml) in Antigen-buffer mix 1 ml/site with antigen in buffer or in the same adjuvant given IM at *=Subcutaneous; IM=Intramuscular

4 CHICKENS: mg of the antigen in up to 1.0 ml total volume of adjuvant-antigen mix. Primary Inoculation: TiterMax & TiterMax Gold /Antigen-buffer (1:1 v/v) - Total volume 1.0 ml, IM (0.1 ml/site at ) and (0.2 ml/site at ) Booster Inoculations: 7 day intervals with antigen in buffer or with the adjuvant, IM, ml/site at until sufficient antibody levels have been reached. Usually this occurs after the third boost. Subsequent boosts may be given periodically to maintain the antibody levels for regular collections. The need for such boosts will be dictated by antibody levels measured in test samples. Pre-bleeds and Test bleeds- (1-2 cc) from the medial metatarsal vein or wing vein. Antibodies will be harvested from yolks of eggs laid. Anesthetics and tranquilizers should not be necessary when collecting blood or egg samples. Product supplied to the investigator will be labeled eggs. ALTERNATIVE ADJUVANT USE in CHICKENS: Complete Freund s Adjuvant**- Incomplete Freund s Adjuvant- Aluminum hydroxide- 100mg Antigen in buffer absorbed to 1mg Aluminum hydroxide. Total volume 0.5 ml IM IM/ IP 1 st inoculation only Booster inoculations 1 site saline or in IFA, given /IM (see next) Boosters given every days. Anticipated that humoral response will be highest by the 3rd boost. *IP=Intraperitoneal; =Subcutaneous; IM=Intramuscular

5 MICE & RATS: An equal volume ( μl) of TiterMax Gold will mixed with approximately μg of recombinant antigen/protein by vortexing. The mixture will be injected, IM or ID into multiple sites (5-8) for the initial injection and subsequent booster injections. A volume of up to 0.5 ml in rats or mice split into SC (up to 0.2ml per site), IM (rats only) (up to 0.1 ml per site) or ID in rats or mice (up to 0.05ml/site) every 10 to 30 days Pre-bleeds and Test bleeds (100 μl) from the tail vein, lateral tarsal vein or submandibular vein. Terminal bleeds will be performed with the animal under general anesthesia (Ketamine 80 mg/kg w/ Xylazine 10 mg/kg IP) via cardiac puncture ALTERNATIVE ADJUVANT USE in MICE and RATS: Complete Freund s Adjuvant**- Antigen/buffer 0.25 /site, max. (1:1 v/v) 1st inoculation only saline or in IFA, given (see IFA below) Incomplete Freund s Adjuvant-Antigen/buffer, (1:1 v/v) Booster inoculations RIBI - Total volume 1.0 ml 0.25 ml/site, at an interval of 3-4 weeks with the antigen in the same adjuvant given with exception for monoclonal Ab may use up to 4 boosts (the 4th boost, only antigen, 5 days prior to euthanasia) Alhydrogel -Antigen/buffer (10:1 to 1:1 v/v) 0.25 ml/site, with the antigen in saline or in the same adjuvant given (0.25 ml/site at ) Saponin (3 mg/ml) in Antigen-buffer mix with antigen in buffer or in the same adjuvant given (0.25 ml/site at ) =Subcutaneous Euthanasia

6 If euthanasia is necessary for maximum blood harvests, or for health and well being of the animal, the following methods will be used: RABBITS: Dose: 1cc (180mg) /3 kg BW GOATS: Dose: 1.5cc/10 lbs body weight SHEEP: Dose: 1.5cc/10 lbs body weight CHICKENS: Dose: 1cc/3kg body weight Mice and rats: Method: CO 2 anesthesia followed by cervical dislocation and exsangination