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SAM 300.03

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United States Department of Agriculture

Center for Veterinary Biologics

Testing Protocol

SAM 300

Supplemental Assay Method for Titration of Neutralizing Antibody

Date: October 23, 2014 Approved, Pending Standard Requirement

Number: SAM 300.03

Supersedes: SAM 300.02, February 11, 2011

Standard Requirement: 

Contact: Alethea M. Fry, (515) 337-7200

Peg A. Patterson

Approvals: /s/Geetha B. Srinivas Date: 22Dec14

Geetha B. Srinivas, Section Leader

Virology

/s/Byron E. Rippke Date: 29Dec14

Byron E. Rippke, Director

Policy, Evaluation, and Licensing

Center for Veterinary Biologics

/s/Rebecca L.W. Hyde Date: 29Dec14

Rebecca L.W. Hyde, Section Leader

Quality Management

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.
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Supplemental Assay Method for Titration of Neutralizing Antibody

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Table of Contents

1. Introduction

2. Materials

2.1 Cell cultures

2.2 Growth medium for cell cultures

2.3 Maintenance medium for cell cultures

2.4 Indicator virus

2.5 Diluent

2.6 Standard reference antiserum

2.7 Test sera

3. Performance of the Test

4. Interpretation of the Test Results

5. Report of Test Results

6. References

7. Summary of Revisions
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Supplemental Assay Method for Titration of Neutralizing Antibody

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1. Introduction

This Supplemental Assay Method (SAM) describes an in vitro assay method for infectious 

canine hepatitis antiserum. The method uses a cell culture system to determine level of antibody 

against infectious canine hepatitis (ICH) viral antigens in commercial antisera.

2. Materials

2.1 Cell cultures

Use secondary (first subculture) or tertiary (second subculture) dog kidney (DK) cells. 

They are grown in roller tubes and are used when approximately 70% confluent. They are 

planted 1.0 mL per tube.

2.2 Growth medium for cell cultures

Gibco’s F-15 medium (Eagles Minimum Essential Medium with Earles salts base, Lglutamine, and nonessential amino acids) is used with 5% fetal calf serum (heated to 

56°C for 30 minutes), 0.5% lactalbumin hydrolysate, 1.0% sodium pyruvate, and 0.22% 

(2.2 gms/l) sodium bicarbonate added. Before adding the serum, the complete medium is 

filtered through a 0.22 micron membrane filter (Millipore Corp.).

Penicillin, 100 IU per mL, and streptomycin, 100 mcg per mL, are used in the medium. 

Antibiotics to control fungi and mycoplasmata may also be used if desired.

2.3 Maintenance medium for cell cultures

This is growth medium with a different serum content. Fetal calf serum (heated at 56°C 

for 30 minutes) is added at the rate of 1% to 5% depending upon growth rate of the cell 

sheet. Two percent is usually optimum.

2.4 Indicator virus

Mirandola strain of infectious canine hepatitis virus (ICH) in the 31st passage in DK cells 

is used as an indicator virus. Batches are prepared as needed, stabilized, and stored frozen 

in appropriate aliquots at -80°F.

2.5 Diluent

Maintenance medium is used to make dilutions of virus and sera.
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2.6 Standard reference antiserum

This is a standard reference supplied by the Center for Veterinary Biologics (CVB).

2.7 Test sera

These are tested at final dilutions of 1:50, 1:150, 1:450, and 1:1350.

3. Performance of the Test

3.1 Dilution of the indicator virus

A geometric mean titer of the indicator virus is determined by at least 3 weekly titrations 

in DK cells. Utilizing this mean titer, the indicator virus is diluted with maintenance 

medium so that 1 mL of diluted virus contains 1,000 to 3,000 TCID50. This will provide 

100 to 300 TCID50 of virus per 0.1 mL, the amount inoculated into each tissue culture 

(TC) tube in the serum neutralization (SN) test system. When calculating the dilution 

factor, aim for 2,000 TCID50 per mL in the working virus. Following are two examples 

for calculating the dilution factor. Assume that the mean titer of the indicator virus is 106

TCID50/mL (1,000,000 TCID50/mL).

Example 1

Arithmetic Method - Divide 1,000,000 by 2,000 (the desired TCID50/mL) to arrive at the 

dilution factor.

1,000,000 = 500, the dilution factor

 2,000

Example 2

Logarithmic Method - Divide 106by 103.3 (103.3 = 2,000, the desired TCID50/mL). 

Division of logarithms is done by subtracting one exponent from the other.

106- 103.3 = 102.7 or 500, the diluent factor

Now that the dilution factor (final dilution of 1:500) has been determined, make serial 

dilutions as follows:

1 mL virus + 4 mL diluent = 1:5 dilution

1 mL of 1:5 dilution + 9 mL diluent = 1:50 dilution

1 mL of 1:50 dilution + 9 mL diluent = 1:500 dilution
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which yields 2,000 TCID50 per mL or 200 TCID50 per 0.1 mL. This dilution of indicator 

virus (200 TCID50/0.1 mL) is referred to as the working virus.

3.2 Standard antiserum

The standard reference infectious canine hepatitis antiserum is tested at the same time, in 

the same cell system, and by exactly the same method as the unknown sera being tested.

3.3 Dilution of test serum

Threefold dilutions are made of the serum to be tested. Final dilutions of 1:50, 1:150, 

1:450, and 1:1350 are tested. The addition of an equal part of working virus doubles the 

dilution, therefore dilutions of 1:25, 1:75, 1:225, and 1:675 of serum only are made. To 

arrive at these dilutions, set up 5 diluent blanks and make serial dilutions as follows:

Add 1 mL of Undiluted Serum

Throughout the dilution procedure, transfers are made with a 1 mL disposable pipette. 

Mixing is done using an eccentric, rotating mixer (Vortex or similar). (Serum 

concentrates must be diluted to the equivalence of unconcentrated serum before testing 

[See Standard Requirement S-28]).

3.3.1 To tube No. 1 add 1 mL of test serum. Discard pipette. Mix. This gives a 

1:5 dilution of serum. 

3.3.2 To tube No. 2 add 1 mL of the mixture from tube No. 1. Discard pipette. 

Mix. This gives a 1:25 dilution.

1 mL of 

serum

1 mL from

Tube #1

4 mL of 

diluent

1 mL from

Tube #2

1 mL from

Tube #3

1 mL from

Tube #4

4 mL of 

diluent

2 mL of 

diluent

2 mL of 

diluent

2 mL of 

diluent

Tube #1

 1:5

Tube #2

 1:25

Tube #3

 1:75

Tube #4

 1:225

Tube #5

 1:675

1 mL 1 mL 1 mL 1 mL
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3.3.3 To tube No. 3 add 1 mL of the mixture from tube No. 2. Discard pipette. 

Mix. This gives a 1:75 dilution.

3.3.4 To tube No. 4 add 1 mL of the mixture from tube No. 3. Discard pipette. 

Mix. This gives a 1:225 dilution.

3.3.5 To tube No. 5 add 1 mL of the mixture from tube No. 4. Discard pipette. 

Mix. This gives a 1:675 dilution.

3.4 Serum neutralization of working virus

Serum/virus mixtures are made by adding an equal volume of the working virus to each 

dilution of the serum on test. Set up 4 tubes each containing 1 mL of a test dilution of 

serum--1:24, 1:75, 1:225, and 1:675. To each tube add 1 mL of working virus. 

 Initial Final

 Serum Working Serum Final Virus

Tube Dilution plus Virus equals Dilution Concentration .

 1 1 mL of 1:25 + 1 mL (2000 TCID50) = 2 mL of 1:50 containing 2000 TCID50

 2 1 mL of 1:75 + 1 mL (2000 TCID50) = 2 mL of 1:150 containing 2000 TCID50

 3 1 mL of 1:225 + 1 mL (2000 TCID50) = 2 mL of 1:450 containing 2000 TCID50

 4 1 mL of 1:675 + 1 mL (2000 TCID50) = 2 mL of 1:1350 containing 2000 TCID50

The serum/virus mixtures are incubated for 1 hour at room temperature before 

inoculating tissue culture tubes.

3.5 Inoculation of roller tubes

Before inoculating the dog kidney monolayers, the growth medium is replaced with 1.5 

mL of maintenance medium. Five cell culture tubes are inoculated with each serum/virus 

mixture (0.2 mL per tube).

3.6 Controls

3.6.1 The indicator virus is titrated to ascertain if titer is being maintained under 

storage conditions.

3.6.2 A 1:2 dilution of the working virus is titrated in tenfold dilutions to 

determine the actual amount of virus used in the SN test. These dilutions are 

incubated along with serum/virus mixtures before inoculation of culture tubes.

3.6.3 Five uninoculated cell culture tubes are incubated with the test to monitor 

the cell system.
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3.6.4 Titration of the standard reference antiserum constitutes a positive serum 

control.

3.7 Incubation

Inoculated and uninoculated cell culture tubes are incubated at 35°- 37°C. Maintenance 

medium is changed every 48 hours regardless of pH change in the medium.

3.8 Reading

Tubes are read daily for typical ICH cytopathogenic effect (CPE) beginning on the 4th 

day and ending on the 10th day postinoculation at which time the test is concluded.

4. Interpretation of the Test Results

The number of tubes in each dilution showing CPE is recorded and the 50% endpoint of 

neutralization (ND50) is calculated by the statistical method of Reed and Muench or SpearmanKärber. 

Note: The ND50 is based upon the dilutions after working virus is added, not on dilutions of 

serums alone.

A serial of antiserum to be considered satisfactory must meet minimums as described in Standard 

Requirement S-28 virus neutralization potency test for ICH antiserum.

5. Report of Test Results

Record all test results on the test record.

6. References

6.1 Robson, D.S.; Hildreth, B.P.; Atkinson, G.F.; Carmichael, L.E.; Barnes, F.D.; 

Pakkala, B.; and Baker, J.A.; Standardization of Quantitative Serological Tests, 65th 

Annual Proceedings, United States Livestock Sanitary Association, October, 1961. Pages 

74-78.

6.2 Carmichael, L.E.; Atkinson, G.F.; and Barnes, F.D.; Conditions Influencing 

Virus-Neutralization Tests for Infectious Canine Hepatitis Antibody, The Corneal 

Veterinarian, Volume LIII, No. 3, July, 1963. Pages 369-388.
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6.3 Herbert, C. Nancy; Stewart, Doreen L.; and Davidson, Ian; International Standard 

for Anti-Canine Hepatitis Serum, Bull. Wld. Hlth. Org., Volume 39, 1968. Pages 909-

916.

6.4 Anonymous: Standard Requirement for Anti-Canine Distemper-HepatitisLeptospira Serum and Products Containing any Combination of These Products. S-28; 

Published by the Veterinary Biologics Division, Agricultural Research Service, USDA, 

January 2, 1964.

6.5 Carmichael, L.E., Factors that Influence the Neutralization Test, 65th Annual 

Proceedings, United States Livestock Sanitary Association, October, 1962. Pages 59-70.

7. Summary of Revisions

Version .03

 The Contact information has been updated; however, the Virology Section has elected 

to keep the same next review date for the document.

Version .02 (February 11, 2011)

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 change was made to the document:

 The Contact information has been updated.