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CVB-DAT-0021.05 Page 1 of 2 

Controlled by: APHIS Center for Veterinary Biologics, 515-337-6100 

United States Department of Agriculture

Animal and Plant Health Inspection Service

Center for Veterinary Biologics

P. O. Box 844

Ames, IA 50010 

1. Reagent Name: Clostridium perfringens Type D (epsilon) Antitoxin 

2. Strain or Source: Not applicable 

3. Lot Number: IRP 249 

4. Fill Date: December 9, 1981 

5. Expiration Date: February 28, 2029 

6. Intended Use: To serve as the standard antitoxin when conducting C. perfringens type D 

(epsilon) toxin-neutralization tests in mice. 

7. Instructions for Use: Clostridium perfringens type D (epsilon) antitoxin IRP 249 

contains 44 antitoxin units per mL (AU/ml). A dilution of standard antitoxin containing 

1.0 AU/mL is used in the toxin-neutralization test as described in title 9, Code of Federal

Regulations (9 CFR), sections 113.112 and 113.455. The dilution may be prepared by adding 2.0 

mL of IRP 249 to 86 mL of peptone diluent. The 1:44 dilution of antitoxin is stable when 

stored at -70°± 10°C and should be aliquoted for future testing. 

8. Test of Reagent: 

Determination of antitoxin titer - The antitoxin titer was determined by injecting mice with 

0.2-mL volumes of diluted antitoxin mixed with 10 L+ toxin doses (the smallest amount of toxin 

which, when mixed with 1.0 unit of antitoxin, causes death in at least 80% of the animals within 

24 hours) and 10 Lo toxin doses (the smallest amount of toxin which, when mixed with 1.0 unit 

of antitoxin, causes no death in animals within 24 hours). The antitoxin titer of IRP 249 was 

confirmed by injecting mice with toxin-antitoxin mixtures that contained 1.0 mL of IRP 249 

diluted 1:44 and mixtures that contained 1.0 mL of C. perfringens type D International antitoxin 

possessing 1.0 unit of antitoxin per mL. 

Sterility test - IRP 249 was tested for sterility by inoculating it in sterile fluid thioglycollate 

medium and streaking it on 5% bovine blood agar plates. No detectable growth appeared in the 

medium or on the blood plates. 

Precautions: There are no known hazards associated with the use of this reagent.
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CVB-DAT-0021.05 Page 2 of 2 

Controlled by: APHIS Center for Veterinary Biologics, 515-337-6100 

9. Container Size, Type, Weight, or Volume: Five-mL glass vials each containing 3.4 mL 

of antitoxin. 

10. Storage Conditions: Store at -25°C or lower. Once the antitoxin has been thawed, store 

at 2o to 7°C. 

11. CVB Technical Contact: Bacteriology Section, Center for Veterinary Biologics, 

(515) 337-6100 or FAX (515) 337-7673. 

12. Origin and Passage History: Not applicable 

13. Method of Preparation: Ponies with no history of clostridial vaccinations received 

multiple injections of C. perfringens type D toxoid and type D toxin during a 12-month period. 

Serums from the hyper-immunized ponies were fractionated with ammonium sulfate and the 

immunoglobulin dialyzed against .015 M phosphate buffered saline, pH 6.8. The antitoxin was 

passed through a Millipore filtration unit containing a 0.22-μm membrane and mixed with 20% 

sterile glycerol. A final concentration of 1:10,000 Thimerosal was added to the glycerinated 

antitoxin. 

14. Other: None 

Reagent orders and feedback should be sent including phone number to the following email 

address: VS.DB.CVB.Reagent.Requests@usda.gov

Reagent orders forms (APHIS Form 2018) can be found on the CVB website.