CONTROLLED//PROPIN//BASIC 

CVB-DAT-0141.04 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 novyi Type B Toxin 

2. Strain or Source: Not applicable

3. Lot Number: IRP 636 

4. Fill Date: October 06, 2016 

5. Expiration Date: February 28, 2029 

6. Intended Use: IRP 636 serves as the standard toxin when conducting C. novyi alpha 

toxin-neutralization tests in mice. 

7. Instructions for Use: IRP 636 diluted 1:30 is considered the standard toxin dilution 

when conducting toxin-neutralization tests in mice as outlined in title 9, Code of Federal

Regulations (9 CFR), section 113.108. The solution of test toxin is prepared by adding 

0.5 mL of well mixed IRP 636 to 14.5 mL of sterile peptone diluent (1.0% peptone, 0.25% 

sodium chloride, (pH 7.2)). A volume of 0.6 mL of the toxin diluted 1:30 and 0.4 mL of diluent 

is equivalent to 0.1 Lo dose. A volume of 0.9 mL of toxin and 0.1 mL of diluent is equivalent to 

0.1 L+ dose.

8. Test of Reagent: Determination of test dose of toxin – The 0.1 Lo and 0.l L+ doses were 

established by injecting mice intravenously with 0.2 ml of a mixture of varying amounts of IRP 

636 combined with 0.1 International Unit of antitoxin. The 0.1 Lo dose for the C. novyi type B 

toxin neutralization test is the largest amount of toxin which can be mixed with one-tenth unit of 

Standard Antitoxin and not cause death in injected mice within 72 hours. The 0.1 L+ dose for the 

C. novyi type B toxin neutralization test is the smallest amount of toxin which can be mixed with 

one-tenth unit of Standard Antitoxin and cause death in at least 80% of injected mice within 72 

hours. 

Determination of toxin type – Toxicity studies in mice demonstrated that the lethal effect of 

C. novyi type B toxin IRP 636 was neutralized by C. novyi type A (alpha) antitoxin. Test results 

showed that IRP 636 contains no detectable beta toxin. 

Precautions: This reagent does not present a hazard to laboratory personnel who work with the 

toxin provided sound fundamental laboratory techniques are followed. 
CONTROLLED//PROPIN//BASIC 

CVB-DAT-0141.04 Page 2 of 2 

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

Sterility test – The toxin was tested for sterility and found to be free of viable bacteria and fungi 

according to procedures outlined in 9 CFR 113.26.

9. Container Size, Type, Weight, or Volume: Freestanding micro tubes (1.5 mL) 

containing 0.7 mL of toxin. 

10. Storage Conditions: Store at -50° to -80°C.

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

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

12. Origin and Passage History: C. novyi type B strain CN 234.3 (IRP 79), used to produce 

IRP 636, was obtained from Wellcome Research Laboratories, Beckenham, England, on July 16, 

1965. The number of passages is unknown.

13. Method of Preparation: Culture CN 234.3 (IRP 79) was grown in dialysis membranes 

with a molecular weight cutoff range from 12,000 to 14,000 daltons. The membranes were filled 

with 0.15 M phosphate buffered saline, pH 7.4, and suspended in 1-liter trypsinizing flasks 

containing media consisting of proteose peptone, trypticase, yeast extract, and liver powder. 

Actively growing culture was aseptically added to the inside of the dialysis membranes and 

incubated at 35oC for 120-140 hours in an anaerobic glove box containing 85% nitrogen (N), 

10% hydrogen (H), and 5% carbon dioxide (CO). The culture was centrifuged at 10,000 x g for 

45-60 minutes. The culture supernatant was passed through a sterile Corning 150-mL bottle top 

filter containing a 0.22-

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.