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SAM 202.06

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United States Department of Agriculture

Center for Veterinary Biologics

Testing Protocol

SAM 202

Supplemental Assay Method for Potency Testing Clostridium perfringens

Type C Beta Antitoxins

Date: December 12, 2017

Number: SAM 202.06

Supersedes: SAM 202.05, June 18, 2014

Standard Requirement: 9 CFR 113.454

Contact: Janet M. Wilson, (515) 337-7245

Approvals: /s/Larry R. Ludemann Date: 19Dec17

Larry R. Ludemann, Section Leader

Bacteriology

/s/Paul J. Hauer Date: 27Dec17

Paul J. Hauer, Director

Policy, Evaluation, and Licensing

Center for Veterinary Biologics

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.

Entered into CVB Quality Management System

by: /s/Linda S. Snavely 27Dec17 

 Linda S. Snavely Date

 Quality Management Program Assistant
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Supplemental Assay Method for Potency Testing Clostridium perfringens Type C Beta Antitoxins

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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/instrumentation

3.3 Preparation of reagents/control procedures

4. Performance of the Test

4.1 Toxin neutralization

4.2 Inoculation of mice

5. Interpretation of the Test Results

5.1 Criteria for a valid test

5.2 Interpretation of serial 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 Antitoxins

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1. Introduction

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

antitoxin content of Clostridium perfringens type C antitoxins as prescribed by the title 9, Code 

of Federal Regulations (9 CFR), part 113.454. The antitoxin 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 Freezer, -70oC

2.1.3 Micropipettes, 100-μL and 1000-μL

2.1.4 Refrigerator, 2o- 7oC

2.1.5 Autoclave

2.2 Reagents/supplies

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

2.2.1 C. perfringens type C (beta) standard antitoxin IRP 637 (available from 

the Center for Veterinary Biologics (CVB))

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

CVB)

2.2.3 Peptone diluent

2.2.4 Tubes, various sizes with lids

2.2.5 Pipettes, various sizes

2.2.6 Syringes, 1-cc, luer-lock
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2.2.7 Needles, 25- to 27-gauge x 1- to 1 1/4-inch

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

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

2.2.10 Tips for micropipettes

2.3 Test animals

White Swiss nonpregnant female mice, 16-20 g (Five mice are required for each toxinantitoxin 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 need specific training in the care and 

handling of laboratory mice.

3.2 Preparation of equipment/instrumentation

All equipment is operated according to manufacturers’ instructions.

3.3 Preparation of reagents/control procedures

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. 

Dispense and autoclave with caps loosened at ≥121°C for 25 to 30 minutes

following manufacturer’s recommendations. Cool flasks and tighten caps. Store 

diluent at 2°- 7°C for up to 3 months.
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3.3.2 Preparation of C. perfringens type C standard beta antitoxin

1. Clostridium perfringens type C antitoxin IRP 637 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. Follow the current Reagent Data Sheet to 

prepare the dilution. 

3.3.3 Preparation of C. perfringens type C standard beta toxin

1. Follow the current Reagent Data Sheet to prepare the dilution of 

C. perfringens type C beta toxin IRP 624. IRP 624, diluted 1:10, is stable 

when stored at -70°± 10°C.

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 (see Sections 4.1.1 and 4.1.2). For the purposes of 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. Performance of the Test

4.1 Toxin neutralization

4.1.1 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 peptone diluent (10 Lo doses)) 

for each product antitoxin dilution and the Lo control using a 17 x 120-mm 

conical tube.
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2. Add 1 mL of each product antitoxin dilution (see table below) to 1 mL

of the standard beta toxin-peptone diluent mixture (10 Lo doses) in 

17 x 100-mm snap-top tubes. Mix each tube with a vortex-type mixer.

Int’l AU 

tested

Unknown Antitoxin 10 Lo doses

Std Toxin Diluent

500 1 mL diluted 1:50

(0.5 mL product + 24.5 mL dil.)

0.5 mL 0.5 mL

600 1 mL diluted 1:60

(0.5 mL product + 29.5 mL dil.)

0.5 mL 0.5 mL

1200 1 mL diluted 1:120

(0.3 mL product + 35.7 mL dil.)

0.5 mL 0.5 mL

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

4. Place tubes in ice.

4.1.2 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 the standard beta toxin-peptone diluent mixture (10 Lo doses) in a 

17 x 100-mm snap-top tube. Mix well with a vortex-type mixer.

2. Add 1.0 mL of standard beta antitoxin containing 10 AU/mL to 0.9

mL of standard beta toxin and 0.1 mL of peptone diluent (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 22o- 26°C for 1 hour.

4. Place tubes in ice.

4.2 Inoculation of mice

4.2.1 Inject 0.2 mL of each standard beta toxin-product antitoxin mixture into 

each of 5 mice.

4.2.2 Inject 0.2 mL of each standard beta toxin-standard beta antitoxin mixture 

into each of 5 mice.

4.2.3 Inoculate all mice intravenously into a lateral tail vein. Use 1-cc needle 

locking syringes fitted with 25- to 27-gauge x 1- to 1 1/4-inch needles. 

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

antitoxin mixtures (controls) last.
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4.2.5 Mouse inoculations should be completed within 1 hour of placing the 

toxin-antitoxin mixtures in the ice.

4.2.6 The test is concluded 24 hours after the mice are inoculated.

5. Interpretation of the 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 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 serial results

5.2.1 The product contains at least 500 International Units of beta antitoxin per 

mL if 5 of the 5 mice inoculated with the 1:50 dilution of product-standard beta

toxin mixture survive.

5.2.2 The product contains at least 600 International Units of beta antitoxin per 

mL if 5 of the 5 mice inoculated with the 1:60 dilution of product-standard beta

toxin survive.

5.2.3 The product is considered unsatisfactory if it contains less than 500 

International Units of beta antitoxin per mL. (If any mice inoculated with the 1:50 

dilution and 10 Lo doses of standard beta toxin die, the product is considered to 

contain less than 500 International Units per mL.)

6. Reporting of Test Results

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

7.1 Title 9, Code of Federal Regulations, part 113.454, 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 637 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 .06

 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 .05

 The Bacteriology Section Leader was updated.

 Minor word changes for clarification of procedures.

Version .04

 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 .03

 The document number has been changed from BBSAM0202 to SAM 202.

 3.3.3: The standard beta toxin use dilution has been adjusted.
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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 119 has changed to IRP 486 throughout the document.

 IRP 418 has changed to IRP 513(04) throughout the document.

 4.1 The format and content have been modified to clarify the LO and L+ levels of the 

Toxin Neutralization process.

 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