Center for Veterinary Biologics SAM 301.05

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Supplemental Assay Method for Titration of Neutralizing Antibody - Canine Distemper

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United States Department of Agriculture

Center for Veterinary Biologics

Testing Protocol

SAM 301

Supplemental Assay Method for Titration of Neutralizing Antibody - Canine 

Distemper

Date: August 22, 2023 Approved, Pending Standard Requirement

Number: SAM 301.05

Supersedes: SAM 301.04, April 21, 2017

Standard Requirement: 

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.
Center for Veterinary Biologics SAM 301.04

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Supplemental Assay Method for Titration of Neutralizing Antibody - Canine Distemper

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Table of Contents

1. Introduction

2. Materials

2.1 Chicken embryos

2.2 Diluent

2.3 Indicator virus

2.4 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 - Canine Distemper

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1. Introduction

This Supplemental Assay Method (SAM) describes a serum neutralization method for assaying 

canine distemper antiserum. The method uses seven-day-old chicken embryos in the test system 

to determine the level of antibody against a measured dose of canine distemper virus.

2. Materials

2.1 Chicken embryos

Seven-day chicken embryos are used in the test system. The embryonated eggs are 

routinely received from the hatchery on the 6th day of incubation and inoculated on the 

7th day. The eggs are candled and all dead or weak embryos are discarded.

2.2 Diluent

Nutrient broth is used to make dilutions of the indicator virus and sera. Difco nutrient 

broth is mixed according to Difco instructions with 200 units of crystalline penicillin G 

and 200 mcg of streptomycin per mL added.

2.3 Indicator virus

The Lederle strain of canine distemper virus in the 40th to 45th egg passage is used as an 

indicator virus. Batches are prepared as needed, stabilized, and stored in the wet state in 

appropriate aliquots at -80°F.

2.4 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 embryos. Utilizing this mean titer, the indicator virus is diluted 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 embryo 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. For these 

examples, assume that the mean titer of the indicator virus is 106 TCID50/mL (1,000,000 

TCID50/mL).
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Supplemental Assay Method for Titration of Neutralizing Antibody - Canine Distemper

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Example 1 

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

dilution factor or final dilution of the virus. 

1,000,000 = 500, the dilution factor 

 2,000 

Example 2 

Logarithmic Method - Divide 106 by 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 

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 canine distemper 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: 
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Supplemental Assay Method for Titration of Neutralizing Antibody - Canine Distemper

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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.)

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.

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. Then to each tube, add 1 mL of working virus. 

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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Supplemental Assay Method for Titration of Neutralizing Antibody - Canine Distemper

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 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 chicken embryos. 

3.5 Inoculation of embryos

The Gorham method is used to inoculate the chorioallantoic membrane (CAM) of the 

embryos. Five embryos are inoculated with each serum/virus mixture (0.2 mL per 

embryo). 

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 eggs. 

3.6.3 Five uninoculated embryos are incubated with the test to monitor the cell 

system.

3.6.4 Titration of the standard reference antiserum constitutes a positive serum 

control. 

3.7 Incubation

Inoculated and uninoculated embryos are incubated at 35°- 37°C. 

3.8 Reading

After 6 days incubation, the eggs are opened and the CAMs examined for white to 

grayish-white plaques usually surrounded by an edematous area. Disregard scar tissue 

caused by needle trauma when CAM was inoculated. 
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Supplemental Assay Method for Titration of Neutralizing Antibody - Canine Distemper

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To open the egg, pierce the shell about 1/2 to 1 inch from the small end with a small pair 

of sharp scissors. Cut through the shell and its membrane in a complete circle. This forms 

a cap of the small end of the egg which when removed exposes the embryo. Discard the 

embryo, leaving the CAM in place. Tease out the CAM with blunt thumb forceps. Place 

all 5 CAMs from the same dilution into a petri dish and examine, using a Quebec counter, 

for typical distemper plaques as described above.

4. Interpretation of the Test Results

The number of CAMs in each dilution showing no plaques is recorded as the number of positive 

responses and the 50% endpoint of neutralization (ND50) is calculated by the statistical method 

of Reed and Muench or Spearman-Kärber. 

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

dilutions alone.

A serial of antiserum to be considered satisfactory must meet or exceed minimums as described 

in Standard Requirement S-28 virus neutralization tests for canine distemper.

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., Factors that Influence the Neutralization Test, 66th Annual 

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

6.3 Gorham, J.R., A Simple Technique for the Inoculation of the Chorioallantoic 

Membrane of Chicken Embryos, Amer. J. Vet. Res., Vol. 18. Pages 691-692

6.4 Steward, D.L.; Hebert, C.N.; and Davidson, I., International Standard for AntiCanine Distemper Serum, Bull. Wld. Hlth. Org., Vol. 39 (1968). Pages 917-924.
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6.5 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. 

6.6 Anonymous: Report of the ad hoc Committee, Standard Reagents and Test 

Procedures, Journal of the AVMA, Vol. 149, September 1, 1966, No. 5, Part 2, Canine 

Distemper Supplement. Pages 717-718. 

7. Summary of Revisions 

Version .05 

• Coversheet and contact information have been updated.

Version .04

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

to keep the same next review date for the document.

Version .03

• The phrase "available from the Center for Veterinary Biologics/CVB" has been 

removed from the document as these reagents are no longer supplied by the CVB. 

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.