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CVB-DAT-0136.05 Page 1 of 3 

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) toxin 

2. Strain or Source: Not applicable 

3. Lot Number: IRP 632 

4. Fill Date: June 2, 2015 

5. Expiration Date: February 28, 2029 

6. Intended Use: IRP 632 serves as the standard toxin when conducting C. perfringens type 

D (epsilon) toxin-neutralization tests in mice. 

7. Instructions for Use: 

Mouse Assay IRP 632 diluted 1:150 is considered the standard toxin dilution when conducting 

toxin-neutralization tests in mice as outlined in 9 CFR 113.112 and 9 CFR 113.455. The dilution 

is prepared by adding 0.5 mL of IRP 632 to 4.5 mL of peptone diluent (1.0% peptone, 0.25% 

sodium chloride, pH 7.2). The toxin is further diluted to 1:150 by adding 1.0 mL of the 1:10 

dilution to 14.0 mL of diluent. A volume of 0.6 mL of the toxin diluted 1:150 and 0.4 mL of 

diluent is equivalent to 10 Lo doses. A volume of 0.9 mL toxin diluted 1:150 and 0.1 mL of 

diluent is equivalent to 10 L+ doses. C. perfringens type D (epsilon) toxin IRP 632 diluted 1:10 

is stable when stored at -60°C or lower. 

MDCK cell assay (Final Product) IRP 632 diluted 1:150 is considered the standard toxin 

dilution when conducting the MDCK cell assay as outlined in CVB-DAT-0008. The dilution is 

prepared by adding 1.0 mL of IRP 632 to 9.0 mL of Minimal Essential Medium (MEM) with 

Earles F-15 with 0.5% LAH, containing 1% L-glutamine and 0.1% pen-strep and 5% fetal 

bovine serum. The toxin is further diluted to 1:150 by adding 1.0 mL of the 1:10 dilution to 14.0 

mL of diluent; this dilution is considered the standard toxin preparation and is used in the toxin 

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

toxin provided sound fundamental laboratory techniques are followed. 
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CVB-DAT-0136.05 Page 2 of 3 

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

control wells. C. perfringens type D (epsilon) toxin IRP 632 diluted 1:10 is stable when stored at 

-60°C or lower. Dilute the standard toxin preparation further according to the following table: 

Add the graduated toxin to each column according to the following template: 

8. Test of Reagent: 

Determination of test dose of toxin The Lo and L+ doses were established by injecting mice 

intravenously with 0.2 mL of a mixture of varying amounts of IRP 632 combined with 0.1 unit 

of standard epsilon antitoxin. The Lo dose for the C. perfringens type D (epsilon) 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 24 hours. The L+ dose 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 24 hours. 

Tube # microliters of 1:150 

toxin 

DMEM in 

mL 

final dilution 

1 154 2 1:2100 

2 182 2 1:1800 

3 222 2 1:1500 

4 286 2 1:1200 

5 400 2 1:900 

 1 2 3 4 5 6 7 8 9 10 11 12 

A Standard Antitoxin Unknown Antitoxin 

B Standard Toxin diluted 1:2100 Standard Toxin diluted 1:1800 Standard Toxin diluted 1:1500 Standard Toxin diluted 1:1200 Standard Toxin diluted 1:900 Live cell Control Standard Toxin diluted 1:1800 Standard Toxin diluted 1:1500 Standard Toxin diluted 1:1200 Standard Toxin diluted 1:900 C 

D 

E Toxin Control F 

G 

H 
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CVB-DAT-0136.05 Page 3 of 3 

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

Determination of LD50 Swiss Webster female mice weighing 16-20 g were injected 

intravenously with 0.2 mL of toxin diluted in peptone diluent. The toxin was found to contain 

104.717 mouse lethal dose fifty (LD50) per mL. 

Determination of toxin type The toxin type was confirmed by performing toxin-neutralization 

tests in mice. The mice were injected intravenously with mixtures of IRP 632 and C. perfringens 

type A, B, C or D antitoxin. All of the mice died within 24 hours except those receiving mixtures 

containing type B or D antitoxin. 

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: 0.8 mL aliquots of toxin in plastic 

microtubes. 

 10. Storage Conditions: Store at -60°C or lower. 

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

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

12. Origin and Passage History: C. perfringens type D (epsilon) culture CN3688, used to 

produce IRP 632, was obtained from Coopers Animal Health, Inc., 1201 Douglas Avenue, 

Kansas City, KS 66103-1438, on January 5, 1976. The number of passages is unknown.

13. Method of Preparation: Culture CN3688 was grown in a 14-liter New Brunswick 

fermentor containing media consisting of N-Z case, proteose peptone, and yeast extract. Actively 

growing culture was aseptically added to the fermentor and incubated at 35°C for approximately 

6 hours. The culture was centrifuged and the supernatant passed through a Millipore filtration 

unit containing a 0.22-

pellicon cassette system containing a high volume ultrafilter. The concentrated toxin was 

adjusted to pH 6.8 and passed through a sterile Millipore filtration unit containing a 0.22-

membrane. Sterile glycerol was added to the product at a final concentration of 5% v/v. 

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.