Center for Veterinary Biologics SAM 203.09

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United States Department of Agriculture

Center for Veterinary Biologics

Testing Protocol

SAM 203

Supplemental Assay Method for Potency Testing Clostridium perfringens

Type D Epsilon Antigen

Date: August 24, 2023

Number: SAM 203

Supersedes: SAM 203.08, May 19, 2023

Standard Requirement: 9 CFR, part 113.112

Contact: Email: Methodsrequest.notification@usda.gov

Phone: Center for Veterinary Biologics, 515-337-6100

United States Department of Agriculture

Animal and Plant Health Inspection Service

P. O. Box 844

Ames, IA 50010

Mention of trademark or proprietary product does not constitute a guarantee or warranty of the product 

by USDA and does not imply its approval to the exclusion of other products that may be suitable.
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Supplemental Assay Method for Potency Testing Clostridium perfringens Type D Epsilon Antigen

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Table of Contents

1. Introduction

2. Materials

2.1 Equipment

2.2 Reagents/supplies

2.3 Test animals

3. Preparation for the Test

3.1 Personnel qualifications/training

3.2 Preparation of equipment and supplies

3.3 Preparation of reagents

4. Performance of the Test

4.1 Vaccination of rabbits

4.2 Collection and preparation of rabbit serum

4.3 Preparation of serum pools

4.4 Toxin neutralization

4.5 Inoculation of mice

5. Interpretation of Test Results

5.1 Criteria for a valid test

5.2 Interpretation of test results

6. Report of Test Results

7. References

8. Summary of Revisions
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1. Introduction

This Supplemental Assay Method (SAM) describes the method used to determine whether 

biological products containing Clostridium perfringens type D epsilon antigen can stimulate the 

production of satisfactory immunity as prescribed by title 9, Code of Federal Regulations

(9 CFR), part 113.112. For products that require 2 vaccinations, rabbits are vaccinated twice 20

to 23 days apart and bled 14 to 17 days following the second vaccination. For products that 

require a single vaccination, rabbits are vaccinated and bled 34 to 40 days later. The serum is 

titrated by a toxin-antitoxin neutralization test using mice as an indicator.

2. Materials

2.1 Equipment

Equivalent equipment may be substituted for any brand name listed below.

2.1.1 Mixer, vortex-type

2.1.2 Centrifuge

2.1.3 Autoclave

2.1.4 Freezers, -20°C and -70°C

2.1.5 Refrigerator, 2°- 7°C

2.1.6 Micropipettes, 100-μL and 1000-μL

2.2 Reagents/supplies

Equivalent reagents or supplies may be substituted for any brand name listed below.

2.2.1 C. perfringens type D epsilon antitoxin, IRP 249

2.2.2 C. perfringens type D epsilon toxin, IRP 632

2.2.3 Peptone diluent

2.2.4 Screw-top Erlenmeyer flask, 500-mL, with cap

2.2.5 Syringes, needle-locking, 1-cc, 10-cc, or 30-cc 

2.2.6 Needles, 25- to 27-gauge x 1/2- to 1 1/4-inch, 20-gauge x 1-inch
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2.2.7 Vacutainer® needles, 20-gauge x 1 1/2-inch

2.2.8 Serum separation tubes, 8.5 - 12.5-mL

2.2.9 Pipettes, 5-mL, 10-mL, 25-mL

2.2.10 Tips for micropipettes

2.2.11 Ketamine hydrochloride, 100 mg/mL solution

2.2.12 Xylazine, 20 mg/mL solution

2.2.13 Water, distilled or deionized, or water of equivalent purity

2.2.14 Glass dilution bottles, 160-mL

2.2.15 Glass screw-cap tubes, 13 x 100-mm

2.2.16 Polystyrene snap-top tubes, 17 x 100-mm with caps

2.2.17 Polystyrene screw-cap tubes, 17 x 120-mm

2.2.18 Polypropylene conical screw-cap tubes, 50-mL

2.3 Test animals

2.3.1 New Zealand White rabbits, non-pregnant females, 4-8 lb (Eight rabbits 

are required per serial to be tested.)

2.3.2 White Swiss non-pregnant female mice, 16-20 g (Five mice are required 

for each toxin-antitoxin mixture.)

3. Preparation for the Test

3.1 Personnel qualifications/training

Technical personnel need a working knowledge of the use of general laboratory 

chemicals, equipment, and glassware and must have specific training and experience in 

the safe handling of clostridial toxins. Personnel must have specific training in the care 

and handling of laboratory rabbits and mice.
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3.2 Preparation of equipment and supplies

3.2.1 Sterilize all glassware before use.

3.2.2 Use only sterile supplies (pipettes, syringes, needles, etc.)

3.2.3 Operate all equipment according to the manufacturers’ instructions.

3.3 Preparation of reagents

3.3.1 Peptone diluent

Peptone (Difco) 8 g

NaCl, reagent grade 2 g

Water q.s. to 800 mL

Dissolve peptone and sodium chloride in water. Adjust pH to 7.2 with 1N sodium 

hydroxide. Fill a 500-mL Erlenmeyer flask no more than 3/4 full with diluent. 

Autoclave with caps loosened at ≥121°C for 25 to 30 minutes following 

manufacturer’s recommendations. Cool flasks and tighten caps. Store at 2°- 7°C 

for up to 3 months.

3.3.2 Preparation of C. perfringens type D standard epsilon antitoxin

1. C. perfringens type D epsilon antitoxin, IRP 249, contains 44 antitoxin 

units per mL (AU/mL) and has been standardized against the World 

Health Organization C. perfringens (C. welchii) type D international 

antitoxin. Each vial contains 3.4 mL of antitoxin.

2. Prepare a dilution of antitoxin that contains 1.0 AU epsilon antitoxin 

per mL by adding 2.0 mL of IRP 249 to 86.0 mL of peptone diluent in a 

160-mL glass dilution bottle. Dispense the diluted antitoxin in 2.5 mL

amounts in 13 x 100-mm screw-cap tubes and store at -70°± 5°C until 

used. For the purpose of this test, the 1.0 AU/mL dilution of antitoxin is 

referred to as the standard epsilon antitoxin.

3.3.3 Preparation of C. perfringens type D epsilon toxin

Prepare a 1:10 dilution of C. perfringens type D epsilon toxin by adding 0.5 mL

of IRP 632 to 4.5 mL of peptone diluent in a screw-cap tube. Dispense diluted 

toxin in 1.5 mL amounts in 13 x 100-mm screw-cap tubes. IRP 632, diluted 1:10, 

is stable when stored at -60°C or lower.
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4. Performance of the Test

4.1 Vaccination of rabbits

4.1.1 Thoroughly shake each bottle of product and wipe the top with alcohol 

before filling the syringe.

4.1.2 Vaccinate each rabbit subcutaneously in the shoulder region with half of 

the largest recommended dose for any species indicated on the product label. Use 

10-, 20-, or 30-cc syringes fitted with 20-gauge x 1-inch needles to vaccinate the 

rabbits.

4.1.3 For products that require 2 vaccinations, give the second vaccination 20 to 

23 days after the first.

4.2 Collection and preparation of rabbit serum

4.2.1 Collect blood from the test rabbits 34 to 40 days after vaccination (or 14 to 

17 days after the second vaccination for products that require 2 vaccinations).

4.2.2 Anesthetize rabbits for bleeding with a mixture of 1.32 mg/kg of xylazine 

and 8.8 mg/kg of ketamine hydrochloride. Give the anesthetic mixture by 

intramuscular injection.

4.2.3 Use 8.5 - 12.5-mL serum separation tubes fitted with a 20-gauge x 1 1/2-

inch Vacutainer® needle to collect blood from the heart. Collect approximately 

10 mL blood from each rabbit. Gently invert tubes 5 times. Let the tubes of blood 

sit at 22°- 26°C (room temperature) for 30 to 60 minutes.

4.2.4 Centrifuge blood at 1000 x g for 10 to 20 minutes at room temperature.

4.3 Preparation of serum pools

4.3.1 Prepare a pooled sample using an equal volume of serum from at least 7 

rabbits per vaccinated group (provided that, if more than 7 rabbits are bled per 

vaccinated group, then equal volumes from each rabbit are used for the serum 

pool). If less than 7 rabbits are bled, the test is invalid and should be repeated. 

4.3.2 The pooled sample may be held at 2°- 7°C for up to 7 days. If testing will 

not be completed within 7 days, store the pooled sample at -20°C or lower. 
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4.3.3 To test for 2 AU/mL of antitoxin, use 1.0 mL pooled serum diluted with 

1.0 mL peptone diluent.

4.3.4 To test for 3 AU/mL of antitoxin, use 1.0 mL pooled serum diluted with 

2.0 mL peptone diluent.

4.4 Toxin neutralization

4.4.1 Preparation of standard epsilon toxin 

1. Further dilute the toxin prepared in Section 3.3.3 to 1:150 by adding 

1.0 mL of diluted (1:10) toxin to 14.0 mL of peptone diluent in a 

17 x 100-mm snap-top tube. For the purpose of this test, the 1:150 dilution 

of IRP 632 is referred to as the standard epsilon toxin.

2. A volume of 0.6 mL standard epsilon toxin and 0.4 mL peptone 

diluent represents 10 Lo doses. A volume of 0.9 mL standard epsilon toxin

and 0.1 mL peptone diluent represents 10 L+ doses.

3. For the purposes of this SAM, 10 Lo dose is defined as the greatest 

amount of toxin that, when mixed with 1.0 AU, results in 100% survival 

of all mice inoculated intravenously (IV) with 0.2 mL of this mixture. The 

10 L+ dose is defined as the least amount of toxin that, when mixed with 

1.0 AU, results in the death of 80% -100% of all mice inoculated IV with 

0.2 mL of this mixture.

4. Mix a sufficient volume of standard epsilon toxin and peptone diluent 

(0.6 mL of standard epsilon toxin and 0.4 mL of peptone diluent (10 Lo

doses) in a 17 x 120-mm conical tube for each serum pool and the Lo

control).

4.4.2 Preparation of standard epsilon antitoxin

Thaw C. perfringens type D epsilon antitoxin, IRP 249 previously diluted 1:44

(see Section 3.3.2). This dilution contains 1 AU/mL and is referred to as the 

standard epsilon antitoxin.

4.4.3 Product and standard epsilon toxin

1. Add 1 mL of each of the serum dilutions (see Section 4.3) to 1 mL of 

this standard epsilon toxin-peptone diluent mixture (see Section 4.4.1(4)) 

in 17 x 100-mm snap-top tubes. Mix each tube well using a vortex-type 

mixer.
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2. Let the mixtures sit at 22°- 26°C (room temperature) for 1 hour.

3. Place tubes in ice.

4.4.4 Standard epsilon toxin and antitoxin controls

1. Add 1.0 mL of standard epsilon antitoxin containing 1.0 AU/mL (see 

Section 4.4.2) to 1 mL of the standard epsilon toxin-peptone diluent 

(10 Lo doses) mixture (see Section 4.4.1(4)) in a 17 x 100-mm snap-top 

tube. Mix well with a vortex-type mixer.

2. Add 1.0 mL of standard epsilon antitoxin containing 1.0 AU/mL (see 

Section 4.4.2) to a mixture containing 0.1 mL of peptone diluent and 

0.9 mL of standard epsilon toxin (10 L+ doses) in a 17 x 100-mm snap-top 

tube. Mix well with a vortex-type mixer.

3. Let the mixtures stand at 22°- 26°C (room temperature) for 1 hour. 

4. Place tubes in ice.

4.5 Inoculation of mice

4.5.1 Inject 0.2 mL of each standard epsilon toxin-product antitoxin mixture 

into each of 5 mice.

4.5.2 Inject 0.2 mL of each standard epsilon toxin-standard epsilon antitoxin 

mixture into each of 5 mice.

4.5.3 Inoculate all mice into a lateral tail vein. Use 1-cc syringes fitted with 25-

to 27-gauge x 1/2- to 1 1/4-inch needles. 

4.5.4 Always inoculate the mice receiving the standard epsilon toxin-standard 

epsilon antitoxin mixtures (controls) last.

4.5.5 Mouse inoculations should be completed within 1 hour of placing the 

toxin-antitoxin mixtures in the ice.

4.5.6 The test is concluded 24 hours after the mice are inoculated.
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5. Interpretation of Test Results

5.1 Criteria for a valid test

5.1.1 All 5 mice inoculated with the standard 10 Lo/1.0 AU control mixture 

must survive.

5.1.2 At least 4 of the 5 mice inoculated with the standard 10 L+/1.0 AU control 

mixture must die.

Note: Moribund animals exhibiting clinical signs consistent with the expected 

disease pathogenesis that are unable to rise or move under their own power 

may be humanely euthanized and considered as deaths as outlined in 9 CFR 

117.4.

5.2 Interpretation of test results

5.2.1 If 5 of the 5 mice inoculated with the 1:2 diluted pooled serum-standard 

epsilon toxin mixture survive, the serum contains at least 2 AU/mL of 

C. perfringens epsilon antitoxin, and the product is satisfactory.

5.2.2 If 5 of the 5 mice inoculated with the 1:3 diluted pooled serum-standard 

epsilon toxin mixture survive, the serum contains at least 3 AU/mL of 

C. perfringens epsilon antitoxin, and the product is satisfactory.

5.2.3 The product is considered unsatisfactory if the serum pool from at least 7 

rabbits contains less than 2 epsilon AU/mL. (If any mice inoculated with the 1:2 

serum dilution and 10 Lo doses of standard epsilon toxin die, the product is 

considered to contain less than 2 AU/mL.)

6. Report of Test Results

Report results of the test(s) as described by standard Section operating procedures.

7. References

7.1 Title 9, Code of Federal Regulations, part 113.112, U.S. Government Printing 

Office, Washington, DC.

7.2 History of toxin: C. perfringens type D epsilon culture CN3688, used to produce 

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

City, Kansas 66103-1438, on January 5, 1976. The number of passages is unknown.
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7.3 History of antitoxin: C. perfringens type D epsilon antitoxin (IRP 249) was 

produced in 1981 at the Center for Veterinary Biologics (CVB) (then part of the National 

Veterinary Services Laboratories (NVSL)), Ames, Iowa. The antitoxin is of equine 

origin. 

8. Summary of Revisions 

Version .09 

• Corrected IRP 249 antitoxin units (AU)/mL throughout the document.

Version .08 

• Updated coversheet and contact information.

• Adjusted 10 L+ dose volume to match reagent data sheet.

• Added serum separation tube size range based on current supply. 

• Minor formatting changes.

Version .07 

• Toxin lot IRP 632 replaces IRP 450 throughout the document.

• The Director was updated on the cover page.

Version .06

• The Bacteriology Section Leader was updated. 

• Minor word changes for clarification of procedures. 

Version .05

• The Contact information has been updated. 

Version .04

• One of this document’s signatories has been changed due to personnel changes. 

Version .03 

This document was revised to clarify the practices currently in use at the Center for Veterinary 

Biologics and to provide additional detail. While no significant changes were made that impact 

the outcome of the test, the following changes were made to the document: 

• Humane endpoint language has been added. 

• Dilution/holding vessel sizes have been added for clarification. 

• The contact person has been changed to Janet M. Wilson. 

Version .02 

• Incorporated the use of IRP 450. 

• 4.4.1.2: Corrected a typographical error.