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SAM 207.08

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United States Department of Agriculture 

Center for Veterinary Biologics 

Testing Protocol 

SAM 207

Supplemental Assay Method for Potency Testing Clostridium novyi Type B 

Alpha Antigen 

Date: December 15, 2017

Number: SAM 207.08

Supersedes: SAM 207.07, June 25, 2014

Standard Requirement: 9 CFR 113.108 

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

Approvals: 

/s/Larry R. Ludemann Date: 20Dec17

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
Center for Veterinary Biologics SAM 207.08

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Supplemental Assay Method for Potency Testing Clostridium novyi Type B Alpha Antigen

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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 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 novyi Type B Alpha Antigen

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1. Introduction

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

biological products containing Clostridium novyi type B alpha antigen can stimulate the 

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

(9 CFR), section 113.108. 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. novyi type B alpha antitoxin, IRP 507 (04) (available from the Center 

for Veterinary Biologics (CVB))

2.2.2 C. novyi type B alpha toxin IRP 636 (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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2.2.6 Needles, 25- to 27-gauge x 7/8- to 1 1/4-inch, 20-gauge x 1-inch 

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

2.2.8 Serum separation tubes, 12.5-mL

2.2.9 Pipettes, 2-mL, 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 Polystyrene snap-top tubes, 17 x 100-mm 

2.2.15 Polystyrene screw-cap conical tubes, 17 x 120-mm 

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

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.).
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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. novyi type B alpha antitoxin 

1. C. novyi type B alpha antitoxin, IRP 507 (04), contains 140 antitoxin 

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

Health Organization gas gangrene (C. novyi) International Antitoxin, 

equine origin. Each vial contains 4.5 mL of antitoxin. 

2. Prepare a solution of C. novyi type B alpha antitoxin that contains 

10 AU/mL by adding 1.0 mL of IRP 507 (04) to 13 mL of peptone diluent 

in a 17 x 100-mm snap-top tube. Dispense in 2.25 mL amounts in glass 13 

x 100-mm screw-cap tubes and store at -70°± 10°C until used. 

3.3.3 Preparation of C. novyi type B alpha toxin 

Each vial of C. novyi type B standard toxin IRP 636 contains 0.7 mL of toxin. 

Store the toxin at -50°to -80°C until used. 

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 not more 

than 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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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 a 12.5-mL serum separation tube fitted with a 20-gauge x 1 1/2-inch 

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

12.5 mL of 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 approximately 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 must be repeated. 

4.3.2 The pooled sample may be held at 2°- 7°C for up to 7 days if the test will 

be conducted within that time. If testing will not be completed within 7 days, store 

the pooled sample at -20°C or lower. 

4.3.3 Use 0.2 mL pooled serum diluted with 0.8 mL peptone diluent to test for 

0.5 AU/mL of antitoxin. 

4.3.4 Use 0.1 mL pooled serum diluted with 0.9 mL peptone diluent to test for 

1.0 AU/ml of antitoxin.

4.4 Toxin neutralization 

4.4.1 Preparation of standard alpha toxin 

1. Dilute the C. novyi type B alpha toxin to 1:30 by adding 0.5 mL of IRP 

636 toxin to 14.5 mL of peptone diluent in a conical screw-top tube. For 

the purpose of this test, the 1:30 dilution of IRP 636 is referred to as the 

standard alpha toxin. 
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2. A volume of 0.5 mL of standard alpha toxin and 0.5 mL of peptone 

diluent represents 0.1 Lo dose. A volume of 0.8 mL of standard alpha toxin 

plus 0.2 mL of peptone diluent represents 0.1 L+ dose. 

3. For the purposes of this SAM, 0.1 Lo dose is defined as the greatest 

amount of toxin that, when mixed with 0.1 AU, results in 100% survival 

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

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

0.1 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 alpha antitoxin 

Thaw the 10 AU/mL C. novyi type B alpha antitoxin previously described (see 

Section 3.3.2). Further dilute the antitoxin to 1 AU/mL by adding 1 mL of the 

well mixed 10 AU/mL antitoxin to 9 mL of diluent in a 17 x 100-mm snap-top 

tube. Finally dilute the antitoxin to 0.1 AU/mL by adding 1 mL of the well mixed 

1 AU/mL antitoxin to 9 mL of diluent in a 17 x 100-mm snap-top tube. For the 

purpose of this test, this 0.1 AU/mL dilution of antitoxin is referred to as the 

standard alpha antitoxin. 

4.4.3 Product and standard alpha toxin 

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

(0.5 mL of standard alpha toxin and 0.5 mL peptone diluent [0.1 Lo dose]) 

for each serum pool and the Lo control in a 17 x 120-mm screw-cap tube. 

Add 1 mL of each of the serum dilutions to 1 mL of this alpha toxinpeptone diluent Lo mixture in 17 x 100-mm snap-top tubes. Mix each tube 

with a vortex-type mixer. 

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

3. Place tubes in ice. 

4.4.4 Standard alpha toxin and antitoxin controls 

1. Add 1.0 mL of standard alpha antitoxin (0.1 AU/mL) to 1 mL of the 

alpha toxin-peptone diluent (0.1 Lo dose) mixture (see Section 4.4.3) in a 

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

2. Add 1.0 mL of standard alpha antitoxin (0.1 AU/mL) to a 17 x 100-

mm snap-top tube containing 0.1 mL peptone diluent and 0.9 mL of 

standard alpha toxin (0.1 L+ dose). Mix well with a vortex-type mixer. 
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3. Let the mixtures sit 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 test toxin-product antitoxin mixture into 

each of 5 mice. 

4.5.2 Inject 0.2 mL of each standard test toxin-standard antitoxin mixture into 

each of 5 mice. 

4.5.3 Inoculate all mice intravenously into 1 of the lateral tail veins. Use 1-cc 

syringes fitted with 25- or 27-gauge x 7/8- to 1 1/4-inch needles. 

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

antitoxin mixtures (controls) last. 

4.5.5 Mouse inoculations need to be completed within 1 hour of placing the 

toxin-antitoxin mixtures in ice. 

4.5.6 The test is concluded 72 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 0.1 Lo/0.1 AU control mixture 

must survive. 

5.1.2 At least 4 of the 5 mice inoculated with the standard 0.1 L+/0.1 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. 
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5.2 Interpretation of test results 

5.2.1 If 5 of the 5 mice inoculated with the 1:5 diluted serum-toxin mixture (see 

Section 4.3.3) survive, the serum contains at least 0.5 AU/mL of C. novyi type B 

alpha antitoxin and the product is satisfactory. 

5.2.2 If 5 of the 5 mice inoculated with the 1:10 diluted serum-toxin mixture 

(see Section 4.3.4) survive, the serum contains at least 1.0 AU/mL of C. novyi 

type B alpha antitoxin. 

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

rabbits contains less than 0.5 AU/mL of C. novyi type B alpha antitoxin. 

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 113.108, U.S. Government Printing 

Office, Washington, DC. 

7.2 History of reagents. C. novyi type B alpha antitoxin IRP 507 (04) was produced in 

goats at the National Veterinary Services Laboratories/Center for Veterinary Biologics in 

Ames, Iowa.

7.3 C. novyi type B alpha toxin (IRP 636) was produced at the Center for Veterinary 

Biologics, Ames, Iowa, in October 2016. The toxin was made from C. novyi type B alpha 

strain CN234.3 (IRP 79). The culture was obtained from Wellcome Research 

Laboratories, Beckenham, England, on July 16, 1965. The number of passages is 

unknown. 

8. Summary of Revisions 

Version .08

 Toxin lot IRP 636 replaces IRP 581 throughout the document.

 The Director was updated on the cover page.
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Version .07

 The Bacteriology Section Leader was updated.

 Minor word changes for clarification of procedures.

Version .06

 C. novyi type B alpha toxin IRP 581 has replaced IRP 425 throughout the document.

Version .05

 The Contact information has been updated.

Version .04

 The standard antitoxin IRP 298 has been changed to IRP 507(4) throughout the 

document. 

 The 0.1 Lo and 0.1 L+ doses have been updated throughout the document. 

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: 

 4.4.3 The wording was changed to add clarity. 

 4.4.4 The wording was changed to add clarity. 

 Humane endpoint language was added. 

 Dilution/holding vessel sizes were added for clarification. 

 The contact person was changed to Janet M. Wilson.