SAM 201.08

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United States Department of Agriculture 

Center for Veterinary Biologics 

Testing Protocol 

SAM 201

Supplemental Assay Method for Potency Testing Clostridium perfringens

Type C Beta Antigen 

Date: August 24, 2023

Number: SAM 201.08

Supersedes: SAM 201.07, December 12, 2017

Standard Requirement: 9 CFR 113.111 

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 C Beta Antigen

Table of Contents

1. Introduction 

2. Materials 

2.1 Equipment/instrumentation

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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Supplemental Assay Method for Potency Testing Clostridium perfringens Type C Beta Antigen

1. Introduction

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

biological products containing Clostridium perfringens type C beta antigen can stimulate the 

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

(9 CFR), part 113.111. 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/instrumentation

Equivalent equipment or instrumentation 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 C standard beta antitoxin, IRP 645 (available from the 

Center for Veterinary Biologics (CVB))

2.2.2 C. perfringens type C standard beta toxin, IRP 624 (available from the 

CVB)

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, 20-cc, or 30-cc 
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Supplemental Assay Method for Potency Testing Clostridium perfringens Type C Beta Antigen

2.2.6 Needles, 25- to 27-gauge x 1- to 1 1/4-inch, 20-gauge x 1 inch 

2.2.7 Vacutainer® needles, 20-gauge x 1 1/2-inch 

2.2.8 Serum separation tubes, 8.0 - 12.5-mL

2.2.9 Pipettes, various sizes

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 Tubes, various sizes with lids

2.3 Test animals

2.3.1 New Zealand White rabbits, nonpregnant females, 4-8 lb (Eight rabbits are 

required per serial to be tested.) 

2.3.2 White Swiss nonpregnant 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. 

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 manufacturer’s instructions. 
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Supplemental Assay Method for Potency Testing Clostridium perfringens Type C Beta Antigen

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. Autoclave at ≥ 121°C for 25 to 30 minutes following manufacturer’s 

recommendations. Store at 2°- 7°C for up to 3 months. 

3.3.2 Preparation of C. perfringens type C standard beta antitoxin 

1. Clostridium perfringens type C beta antitoxin IRP 645 contains 550

units of beta antitoxin per mL (AU/mL) and has been standardized against 

the World Health Organization C. perfringens (C. welchii) type C 

International antitoxin. Each vial contains 0.6 mL of antitoxin. 

2. A dilution of standard beta antitoxin containing 10 AU/mL is used in 

the toxin-neutralization test. To prepare the initial dilution, add 0.5 mL of 

well mixed IRP 645 to 4.5 mL of peptone diluent. Dispense and store 

aliquots of IRP 645, diluted 1:10. This dilution contains 55 AU/mL and is 

stable when stored at -50° to -80°C. 

3.3.3 Preparation of C. perfringens type C beta toxin 

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

IRP 624 to 4.5 mL of peptone diluent. Dispense and store aliquots of IRP 624, 

diluted 1:10. This dilution is stable when stored at -70°± 10°C. 

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. 
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Supplemental Assay Method for Potency Testing Clostridium perfringens Type C Beta Antigen

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 8.0 - 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 

8.0 - 12.5 mL of blood from each rabbit. Gently invert tubes 5 times. Let the tubes 

of blood sit at 20°- 26°C for 30 to 60 minutes. 

4.2.4 Centrifuge blood at 1000 x g for 10 to 20 minutes at 20°- 26°C. 

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. 

4.3.3 Use 1.0 mL pooled serum to test for 10 AU/mL of antitoxin. 

4.3.4 Dilute 1.0 mL pooled serum with 0.2 mL peptone diluent to test for 

12 AU/mL of antitoxin. 

4.4 Toxin neutralization

4.4.1 Preparation of C. perfringens type C standard beta toxin 

Further dilute the C. perfringens type C beta toxin to 1:75 by adding 1.0 mL of 

diluted (1:10) toxin (see Section 3.3.3) to 6.5 mL of peptone diluent. For the 

purpose of this test, the 1:75 dilution of IRP 624 is referred to as the standard beta 

toxin. 

Note: A volume of 0.5 mL of standard beta toxin and 0.5 mL of peptone 

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

and 0.1 mL of peptone diluent represents 10 L+ doses. For the purposes of 
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Supplemental Assay Method for Potency Testing Clostridium perfringens Type C Beta Antigen

this SAM, 10 Lo dose is defined as the greatest amount of toxin that, when 

mixed with 10 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 10 AU, results in the death of 

80%-100% of all mice inoculated IV with 0.2 mL of this mixture. 

4.4.2 Preparation of standard beta antitoxin 

Thaw the C. perfringens type C beta antitoxin IRP 645, previously diluted 1:10 

(see Section 3.3.2). Further dilute the antitoxin to 1:55 by adding 1.0 mL of 

diluted antitoxin (1:10) to 4.5 mL of diluent. This dilution contains 10 AU/mL

and is referred to as the standard beta antitoxin. 

4.4.3 Product and standard beta toxin 

1. Mix a sufficient volume of standard beta toxin and peptone diluent 

(0.5 mL of standard beta toxin and 0.5 mL of peptone diluent (10 Lo

doses)) for each serum pool and the Lo control. Add 1.0 mL of each serum 

dilutions to 1.0 mL of this standard beta toxin-peptone diluent mixture. 

Mix each tube well using a vortex-type mixer. 

2. Let the mixtures stand at 20°- 26°C for 1 hour. 

3. Place tubes in ice. 

4.4.4 Standard beta toxin and standard beta antitoxin controls 

1. Add 1.0 mL of standard beta antitoxin containing 10 AU/mL to 1.0 

mL of standard beta toxin-peptone diluent mixture (10 Lo doses). Mix well 

with a vortex-type mixer. 

2. Add 1.0 mL of standard beta antitoxin containing 10 AU/mL to a 

tube containing 0.9 mL of standard beta toxin and 0.1 mL of peptone 

diluent (10 L+ doses). Mix well with a vortex-type mixer. 

3. Let the mixtures stand at 20°- 26°C for 1 hour. 

4. Place tubes in ice. 

4.5 Inoculation of mice 

4.5.1 Inject 0.2 mL of each standard beta toxin-product serum mixture into each 

of 5 mice. 
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Supplemental Assay Method for Potency Testing Clostridium perfringens Type C Beta Antigen

4.5.2 Inject 0.2 mL of each standard beta toxin-standard beta 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- to 1 1/4-inch needles. 

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

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. 

5. Interpretation of Test Results

5.1 Criteria for a valid test 

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

survive. 

5.1.2 At least 4 of the 5 of the mice inoculated with the standard 10 L+/10 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 undiluted pooled serum-standard 

toxin mixture survive, the serum contains at least 10 AU/mL of C. perfringens

beta antitoxin, and the product is satisfactory.

5.2.2 If 5 of the 5 mice inoculated with the diluted (1.0 mL serum + 0.2 mL

diluent) pooled serum-standard toxin mixture survive, the serum contains at least 

12 AU/mL of C. perfringens beta 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 10 beta AU/mL. (If any mice inoculated with the 

undiluted pooled serum and 10 Lo doses of standard beta toxin die, the product is 

considered to contain less than 10 AU/mL.) 
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Supplemental Assay Method for Potency Testing Clostridium perfringens Type C Beta Antigen

6. Report of Test Results 

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

7. References 

7.1 Title 9, Code of Federal Regulations, part 113.111, U.S. Government Printing 

Office, Washington, DC. 

7.2 History of toxin: C. perfringens type C culture #4414, used to produce IRP 624, 

and was obtained from Coopers Animal Health, Inc., 1201 Douglas Avenue, 

Kansas City, Kansas, on July 28, 1975. The number of passages is unknown. 

7.3 History of antitoxin: C. perfringens type C (beta) antitoxin IRP 645 was produced 

in goats hyperimmunized with multiple injections of purified C. perfringens type C 

toxoid and toxin during a 6-month period. 

8. Summary of Revisions 

Version .08 

• Coversheet information updated.

• Antitoxin lot IRP 645 replaces IRP 637 throughout the document.

Version .07 

• Antitoxin lot IRP 637 replaces IRP 585-A throughout the document. 

• Toxin lot IRP 624 replaces IRP 513 (04) throughout the document. 

• The Director was updated on the cover page. 

Version .06

• The Bacteriology Section Leader was updated.

• IRP 585 has changed to IRP 585-A throughout the document. 

• Minor word changes for clarification of procedures. 

Version .05

• The Contact information has been updated. 

• Standard beta antitoxin lot IRP 585 has replaced IRP 486 throughout the document. 

Due to change in the reagent lot, the use dilution has also changed. 

Version .04 

• 4.4.1 The standard beta toxin use dilution has been adjusted. 

Version .03

• 4.4.2 Typographical errors have been corrected. 

• Dilution/holding vessel specifications throughout the document have been removed to 
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Supplemental Assay Method for Potency Testing Clostridium perfringens Type C Beta Antigen

allow for variations. 

• The ultra-low freezer and room temperature parameters have been adjusted. 

Version .02 

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: 

• IRP 448 has changed to IRP 486 throughout the document. 

• IRP 447 has changed to IRP 513(04) throughout 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.