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SAM 217.07

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United States Department of Agriculture

Center for Veterinary Biologics

Testing Protocol

SAM 217

Supplemental Assay Method for Potency Testing of Tetanus Toxoid by ELISA

Date: February 17, 2017

Number: SAM 217.07

Supersedes: SAM 217.06, March 21, 2014

Standard Requirement: 9 CFR 113.114

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

Approvals: /s/Larry R. Ludemann Date: 06Mar17

Larry R. Ludemann, Section Leader

Bacteriology 

/s/Paul J. Hauer Date: 06Mar17

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 07Mar17 

 Linda S. Snavely Date

 Quality Management Program Assistant
Center for Veterinary Biologics SAM 217.07

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Supplemental Assay Method for Potency Testing of Tetanus Toxoid by ELISA

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Table of Contents

1. Introduction

2. Materials

2.1 Equipment/instrumentation

2.2 Supplies

2.3 Chemicals and reagents

2.4 Test animals

3. Preparation for the Test

3.1 Personnel qualifications/training

3.2 Preparation of equipment/supplies

3.3 Preparation of reagents

4. Performance of the Test

4.1 Vaccination of test animals

4.2 Blood sample collection and serum preparation

4.3 ELISA procedure

4.4 Calculation of results

5. Interpretation of the Test Results

6. Report of Test Results

7. References

8. Summary of Revisions
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Supplemental Assay Method for Potency Testing of Tetanus Toxoid by ELISA

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1. Introduction

This Supplemental Assay Method (SAM) is used to determine the potency of tetanus toxoid as 

prescribed by title 9, Code of Federal Regulations (9 CFR), section 113.114. Guinea pigs are 

vaccinated and bled 6 weeks later. The serum is assayed for antitoxin content by indirect 

Enzyme-Linked Immunosorbent Assay (ELISA).

2. Materials

2.1 Equipment/instrumentation

Equivalent equipment or instrumentation may be substituted for any brand name listed 

below.

2.1.1 Orbital shaker

2.1.2 Test tube mixer, vortex-type

2.1.3 Incubator, 35°- 37°C, with cooling capacity

2.1.4 Refrigerator, 2°- 7°C

2.1.5 Freezer, -20°C or lower

2.1.6 Centrifuge with horizontal centrifuge head that accommodates blood 

collection tubes 

2.1.7 Microplate washer/aspirator

2.1.8 Microplate reader

2.1.9 Computer with software that performs linear regression analysis

2.1.10 Micropipettors, 20-μL, 100-μL, and 1000-μL

2.1.11 Multichannel micropipettor, 50- to 300-μL

2.2 Supplies

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

2.2.1 Tips for micropipettors
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2.2.2 Flat-bottom 96-well microtitration plates (NuncTM F96 maxisorb)

2.2.3 Syringes, needle-locking

2.2.4 Needles, 23-gauge and gauge approved for blood collection

2.2.5 Blood collection tubes

2.2.6 Pipettes, assorted sizes

2.2.7 Tube and flasks (with caps), various sizes to make reagent dilutions

2.2.8 Humidified chamber (small pan with lid or equivalent container with wet 

Kemwipes or paper towels)

2.3 Chemicals and reagents

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

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

2.3.2 Sodium chloride (NaCl)

2.3.3 Nonfat dry milk

2.3.4 Sodium bicarbonate (NaHCO3)

2.3.5 Sodium carbonate (Na2CO3)

2.3.6 Sodium phosphate, dibasic, anhydrous (Na2HPO4)

2.3.7 Sodium phosphate, monobasic, monohydrate (NaH2PO4•H2O)

2.3.8 Sodium hydroxide (NaOH), 5M

CAUTION! Toxic and Hazard Review (THR) of liquid sodium hydroxide: 

Poison by intraperitoneal route. Moderately toxic by ingestion. Mutagenic 

data. A corrosive irritant to skin, eyes, and mucous membranes. This 

material, both solid and in solution, has markedly corrosive action upon all 

body tissue causing burns and frequently deep ulceration, with ultimate 

scarring. Mist, vapors, and dusts of the compound cause small burns, and 

contact with the eyes rapidly causes severe damage to the delicate tissue.
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2.3.9 Polyoxyethylene-sorbitan monolaurate (Tween 20)

2.3.10 Goat anti-guinea pig IgG (H+L) conjugated to horseradish peroxidase, 

Jackson Immunoresearch Laboratories (Catalog #106-035-003)

2.3.11 2,2’-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) 2-

Component Microwell Peroxidase Substrate, SeraCare Life Sciences (Catalog 

#5120-0032 [previously Kirkegaard and Perry Laboratories, Inc. (Catalog #50-62-

00)]

2.3.12 Clostridium tetani toxin, current lot, provided by the Center for Veterinary 

Biologics (CVB)

2.3.13 C. tetani-negative guinea pig serum, current lot, provided by the CVB

2.3.14 C. tetani-positive guinea pig serum, (5 AU/mL), current lot, provided by 

the CVB

2.4 Test animals

Guinea pigs, 450-550 g. A group of 10 guinea pigs consisting of an equal number of 

males and females are required for each serial. Select guinea pigs that are healthy, free of 

external parasites, and have an unblemished hair coat. Do not use pregnant guinea pigs.

3. Preparation for the Test

3.1 Personnel qualifications/training

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

chemicals, equipment, and glassware; and have specific training and experience in the 

safe handling of clostridial toxins. Personnel need specific training in the care and 

handling of laboratory guinea pigs.

3.2 Preparation of equipment/supplies

3.2.1 Operate all equipment according to the manufacturers’ instructions.

3.2.2 Use sterile supplies.
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3.3 Preparation of reagents

3.3.1 Wash solution (National Centers for Veterinary Biologics (NCAH) Media 

#30063)

NaCl 8.5 g

NaH2PO4•H2O 0.22 g 

Na2HPO4 1.19 g

Tween 20 0.5 mL

Deionized water 1.0 L

Mix until completely dissolved. If necessary, adjust the pH to 7.2 with 

5 M NaOH. Autoclave at ≥ 121°C for 30 to 35 minutes following 

manufacturer’s recommendations. Store at 20°- 25°C for up to 6 months.

3.3.2 Antigen coating buffer (NCAH Media #20034)

Na2CO3 0.795 g

NaHCO3 1.465 g

Deionized water 500 mL

Mix until completely dissolved. If necessary, adjust the pH to 9.6 with 5 

M NaOH. Store the buffer at 2°- 7°C for no longer than 1 week.

3.3.3 Blocking solution

Nonfat dry milk 2.5 g

Wash solution (Section 3.3.1) 250 mL

Mix until dissolved. If necessary, heat just enough to dissolve milk. Adjust 

the pH to 7.9 with 5 M NaOH. Store the solution at 2°- 7°C for no longer 

than 1 week.

3.3.4 1% Skim Milk (SM) diluent

Nonfat dry milk 5.0 g

Wash solution (Section 3.3.1) 500 mL

Mix until completely dissolved, heating if necessary. Add 0.25 mL of 

Tween 20 and stir until evenly distributed in the mixture. Adjust the pH to 

7.2 with 5 M NaOH. Store the diluent at 2°- 7°C for no longer than 1 

week.
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4. Performance of the Test

4.1 Vaccination of test animals

4.1.1 Check the container label and the manufacturer’s Outline of Production of 

each product to be tested for identity and recommended field dose. The dose for 

guinea pigs is 0.4 of the largest dose recommended on the product label.

4.1.2 Mix the product thoroughly before filling syringes. Vaccinate each guinea 

pig subcutaneously (using a 23-gauge needle) in the ventral thoracic area.

4.2 Blood sample collection and serum preparation

4.2.1 Collect a 10 mL blood sample from each guinea pig 42 to 45 days after 

vaccination. Use a blood collection protocol approved by the Institutional Animal 

Care and Use Committee. 

4.2.2 Centrifuge the blood from each animal separately at 1000 x g (~2200 

rpm), and dispense the serum into individually marked tubes.

4.2.3 Pool equal portions of serum, but not less than 0.5 mL from each guinea 

pig, and store it at 2°- 7°C for up to 7 days. The serum pool must be made from 

all guinea pigs that survive the vaccination and must contain serum from at least 8 

animals. If the test will not be conducted for at least 7 days after the date of blood 

collection, store serum at -20°C or lower prior to use.

4.3 ELISA procedure

4.3.1 Coat the microtiter plate with tetanus toxin

1. Dilute C. tetani toxin in antigen coating buffer to the working dilution 

specified in the reagent data sheet for the lot of toxin being used.

CAUTION! Accidental parenteral inoculation and ingestion of tetanus 

toxin are the primary hazards to laboratory personnel. It is unknown if the 

toxin can be absorbed through mucous membranes; consequently, the 

hazards associated with aerosols and droplets remain unclear.

2. Dispense 100 μL of diluted toxin into each well of a 96-well 

microtitration plate. Cover the plate. Incubate the plate overnight in a 

humidified chamber at 20°- 25°C on an orbital shaker operating at 50-70 

revolutions per minute (rpm). 
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3. At the end of the incubation period, invert the plate to empty any 

unbound toxin into a discard container and blot on absorbent disposable 

toweling to remove any residual toxin. Autoclave all toxin reagent prior to 

disposal.

4.3.2 Block the microtitration plate 

1. Fill each well of the microtitration plate with 250-300 μL blocking 

solution.

2. Cover the plate and incubate it in a humid atmosphere at 35°- 37°C for 

120 to 140 minutes while mixing on an orbital shaker (50-70 rpm).

Note: Use an incubator with refrigeration capability or the heat from 

the shaker will cause the temperature to increase to an unacceptable 

level.

3. Invert the plate and blot on absorbent toweling to remove the excess 

blocking solution. If not proceeding directly to Section 4.3.3, cover the

plate and place it in a plastic bag. Store the plate in an inverted position at 

2°- 7°C for no longer than 1 week.

4.3.3 Add the serum samples

1. Wash the plate 5 times using 250-300 μL of wash solution per well. 

Invert the plate and tap it on absorbent material to remove all traces of 

residual wash solution.

2. Make an initial 1:4000 dilution of all sera (positive control, negative 

control, and each test serum pool) in SM diluent as shown in Figure 1 and 

Figure 2. This working dilution falls within the dynamic response range 

of the assay.

3. Make additional incremental dilutions of the positive guinea pig serum 

as illustrated in Figure 1. These dilutions will be used to create a standard 

response curve against which the test sera are compared.

4. Add 100 μL of SM diluent to each well in column 1. Use these wells 

to blank the microplate reader. Do not use the remaining outside wells 

because they may provide inconsistent readings.
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1:100

1:100

1:100

5. Add 100 μL of each diluted control serum to each of 3 replicate wells. 

Add 100 μL of each test serum pool to each of 6 replicate wells. An 

example plate diagram is shown in Figure 3.

6. Cover the plate and incubate it in a humid atmosphere for 16 to 24 

hours at 2°- 7°C while mixing on an orbital shaker (50-70 rpm).

Figure 1. 

Dilution of the Positive Guinea Pig Serum and Negative Guinea Pig Serum

0.1 mL

Positive Control

9.9 mL 

SM

1:4000* 

5.0 AU

4.0 AU 3.0 AU 2.0 AU 1.0 AU

Negative Control

0.4 mL

Negativ

e 

guinea 

pig 

Serum

Std. 

Antitoxin

 15.6 mL

 SM

2.0 mL

SM

 3.0 mL 

SM

4.0 mL

SM

0.1 mL

 1.0 mL

 SM

 9.9 mL 

SM

 15.6 mL

 SM

0.4 mL

1:4000* 

0 AU

*Positive guinea pig serum diluted 1:4000 represents 5.0 AU/mL on the standard curve. Negative serum 

represents 0 AU/mL

4.0 mL

3.0 mL

2.0 mL

1.0 mL
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Figure 2. Dilution of the Unknown Guinea Pig Serum

Figure 3. Example Plate Diagram

4.3.4 Add the conjugate

1. Wash the plate as described in Section 4.3.3(1).

2. Dilute the conjugate with SM diluent to the optimized working 

dilution for the lot (1:10,000 is suggested if that dilution falls within the 

manufacturer’s recommended ELISA range). 

3. Add 100 μL of diluted conjugate to each well of the plate.

4. Cover the plate and incubate it in a humid atmosphere at 35°- 37°C for 

120 to 140 minutes while mixing on an orbital shaker (50-70 rpm).

A

B

C

D

E

F

G

H

 Standards Samples

N

E

G

1

A

U

2

A

U

3

A

U

4

A

U

5

A

U

T

E

S

T

S

E

R

U

M

T

E

S

T

S

E

R

U

M

T

E

S

T

S

E

R

U

M

T

E

S

T

S

E

R

U

M

T

E

S

T

R

U

M

T

E

S

T

S

E

R

U

M

T

E

S

T

S

E

R

U

M

S

E

D

I

L

U

E

N

T

B

L

A

N

K

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

0.1 mL 0.4 mL

Unknown 

guinea pig 

Serum

 9.9 mL 

SM

 15.6 mL

 SM

1:4000* 

0 AU

1:100
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4.3.5 Add the substrate

1. Ten to 25 minutes before use, allow the substrate to warm to

20°- 25°C. Protect it from light.

2. Follow the manufacturer’s instructions to prepare 12 mL of ABTS 

substrate. 

3. Wash the plate as described in Section 4.3.3(1).

4. Dispense 100 μL of substrate solution into the wells of the plate.

5. Cover the plate and incubate it at 20°- 25°C for 15 to 90 minutes while 

mixing on an orbital shaker (50-70 rpm). Ideally, the incubation should be 

terminated when the absorbance of the wells containing the 5 AU/mL 

standard is 0.7-1.0.

4.3.6 Read the test plate

Read the absorbance of the ELISA plate at 405/490 nm. 

4.4 Calculation of results

4.4.1 Perform a linear regression analysis using data (antitoxin unit 

concentration vs. absorbance) from the control sera (0-5 AU/mL). Determine the 

slope, correlation coefficient, and y-intercept of the resultant dose-response line 

(i.e., standard curve). 

4.4.2 Calculate the potency (AU/mL) of the test serum pool by fitting the 

average absorbance of the test serum pool to the line defined in Section 4.4.1.

Titer (AU/mL) of test serum pool = Absorbance of test serum pool - y intercept

 Slope

5. Interpretation of the Test Results

5.1 For a valid test, a correlation coefficient of at least 0.985 and a slope of at least 

0.1 must be achieved. Invalid tests may be repeated.

5.2 If the antitoxin titer of the serum pool is at least 2.0 AU/mL, the test serial is 

satisfactory. 

5.3 If the antitoxin titer of the serum pool is < 2.0 AU/mL and the test serial is not 

retested, it shall be declared unsatisfactory. For serials in which the serum pool contains 
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< 2.0 AU per mL, the individual component sera from the pool may be tested by the 

assay described above, using the following criteria:

5.3.1 If at least 80% of a minimum of 8 individual serums have an antitoxin titer 

of at least 2.0 AU per mL, the serial is satisfactory. 

5.3.2 If fewer than 80% of the 8 or more individual serums have an antitoxin 

titer of at least 2.0 AU/mL, the serial may be retested in 10 guinea pigs using the 

same procedure utilized in the initial test. The antitoxin titer of the pooled serum 

from the guinea pigs used in the retest shall be averaged with the antitoxin level 

of the pooled serum from the initial test. If the average of the 2 pools is at least 

2.0 AU/mL, the serial is satisfactory. If the average of the 2 pools is < 2.0 

AU/mL, the serial is unsatisfactory and shall not be retested.

6. Report of Test Results

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

7. References

Title 9, Code of Federal Regulations (9 CFR), section 113.114, U.S. Government Printing 

Office, Washington, DC.

8. Summary of Revisions

Version .07

 Updated ABTS supplier information.

 Increased substrate incubation time and clarified 5 AU/mL control serum readings.

Version .06

 Bacteriology Section Leader updated.

 Minor word changes for clarification of procedures.

Version .05

 The Contact information has been updated.
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 All references to National Veterinary Services Laboratories media have been changed 

to National Centers for Animal Health media.

Version .04

 4.3.5: A wider range of time for the substrate incubation has been added. 

Version .03

 3.3 This section has been reformatted and rewritten for clarity. 

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:

 3.3 This section has been rewritten for clarity.

 3.4 This section has been deleted and the information is covered in other sections of 

the document.

 4.3.3 This section has been rewritten for clarity.

 4.4 This section has been rewritten for clarity.

 5.1 Validity requirement for slope was added.

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