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SAM 302.07

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United States Department of Agriculture

Center for Veterinary Biologics

Testing Protocol

SAM 302

Supplemental Assay Method for Titration of Canine Distemper Virus in 

Primary Cell Culture

Date: October 29, 2014

Number: SAM 302.07

Supersedes: SAM 302.06, September 9, 2011

Standard Requirement: 9 CFR 113.306

Contact: Sandra K. Conrad, (515) 337-7200

Peg A. Patterson

Approvals: /s/Geetha B. Srinivas Date: 31Dec14

Geetha B. Srinivas, Section Leader

Virology

/s/Byron E. Rippke Date: 06Jan15

Byron E. Rippke, Director

Policy, Evaluation, and Licensing

Center for Veterinary Biologics

/s/Rebecca L.W. Hyde Date: 09Jan15

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 Canine Distemper Virus in Primary Cell Culture

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Table of Contents

1. Introduction

2. Materials

2.1 Equipment/instrumentation

2.2 Reagents/supplies

3. Preparation for the Test

3.1 Personnel qualifications/training

3.2 Preparation of equipment/instrumentation

3.3 Preparation of reagents/control procedures

3.4 Preparation of the sample

4. Performance of the Test

5. Interpretation of the Test Results - Valid Assay

6. Report of Test Results

7. References 

8. Summary of Revisions
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Supplemental Assay Method for Titration of Canine Distemper Virus in Primary Cell Culture

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1. Introduction

This Supplemental Assay Method (SAM) describes an in vitro test method for assaying the viral 

content of modified-live canine distemper virus (CDV) vaccines. The test is applicable to CDV 

vaccines when the master seed virus (MSV) is not distinctly cytopathogenic. A direct fluorescent 

antibody (FA) or indirect fluorescent antibody (IFA) staining method is used, utilizing primary 

dog kidney (DKp) cell culture. 

2. Materials

2.1 Equipment/instrumentation

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

below.

2.1.1 36°± 2°C, high humidity, 5% ± 1% CO2 incubator (Model 3336, Forma 

Scientific Inc.)

2.1.2 36°± 2°C aerobic incubator (Model 2, Precision Scientific)

2.1.3 36°± 2°C water bath 

2.1.4 Ultraviolet (UV) light microscope (Model BH2, Olympus America Inc.)

2.1.5 Vortex mixer (Vortex-2 Genie, Model G-560, Scientific Industries Inc.)

2.1.6 Microscope slide glass staining dish with rack (glass staining dish)

2.1.7 Self-refilling repetitive syringe, 2-mL

2.1.8 Pipette-aid

2.1.9 Pipettor with tips and/or motorized microliter pipette and tips

2.2 Reagents/supplies

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

reagents and supplies must be sterile.

2.2.1 CDV Positive Control, Rockborn strain

2.2.2 Monospecific antisera, free of CDV antibody, that neutralize the non-CDV 

fractions present in multifraction vaccines, e.g., canine adenovirus (CAV), canine 

parainfluenza virus (CPI), canine parvovirus (CPV), etc.
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2.2.3 DKp cell culture, free of extraneous agents as tested by title 9, Code of

Federal Regulations (9 CFR), (available from the Center for Veterinary Biologics

(CVB))

2.2.4 For FA: Anti-CDV fluorescein isothiocyanate (FITC)-labeled conjugate 

(Anti-CDV Conjugate) or for IFA: CDV antiserum and anti-dog IgG FITC 

conjugate (Anti-dog Conjugate)

2.2.5 Minimum essential medium (MEM)

1. 9.61 g MEM with Earles salts without bicarbonate

2. 1.1 g sodium bicarbonate

3. Dissolve with 900 mL deionized water (DI).

4. Add 5.0 g lactalbumin hydrolysate or edamine to 10 mL DI. Heat to 

60°± 2°C until dissolved. Then add to the solution in Step 3 with constant 

mixing.

5. Q.S. to 1000 mL with DI; adjust pH to 6.8-6.9 with 2N hydrochloric 

acid (HCl).

6. Sterilize through a 0.22-μm filter.

7. Aseptically add 50 μg/mL gentamicin sulfate

8. Store at 2°- 7°C.

2.2.6 Growth Medium

1. 900 mL MEM

2. Aseptically add:

a. 100 mL gamma-irradiated fetal bovine serum (FBS)

b. 10 mL L-glutamine (200 mM)

3. Store at 2°- 7°C.
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2.2.7 Dulbecco’s phosphate buffered saline (DPBS) (NCAH Media #30040)

1. 8.0 g sodium chloride (NaCl)

2. 0.2 g potassium chloride (KCl)

3. 0.2 g potassium phosphate, monobasic, anhydrous (KH2PO4)

4. 0.1 g magnesium chloride, hexahydrate (MgCl2•6H2O)

5. Dissolve reagents in Steps 1 through 4 with 900 mL DI.

6. Add 1.03 g sodium phosphate, dibasic, anhydrous (Na2HPO4) to 10 

mL DI, heat to 60°± 2°C until dissolved, then add to Step 5 with constant 

mixing.

7. Dissolve 0.1 g calcium chloride, anhydrous (CaCl2) with 10 mL DI

and add slowly to Step 6 to avoid precipitation.

8. Q.S. to 1000 mL with DI; adjust pH to 7.0-7.3 with 2N HCl.

9. Sterilize through a 0.22-μm filter.

10. Store at 2°- 7°C.

2.2.8 Glass slides, 8-chamber (Lab-Tek®Slides) 

2.2.9 Polystyrene tubes, 12 x 75-mm 

2.2.10 Acetone, 100%

2.2.11 Pipettes

2.2.12 Disposable transfer pipette, 3.5-mL

2.2.13 Needles, 18-gauge x 1 1/2-inch

2.2.14 Syringe (tuberculin slip tip), 1-mL
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3. Preparation for the Test

3.1 Personnel qualifications/training

Personnel shall have experience in the preparation and maintenance of cell culture as well 

as in the propagation and maintenance of animal viruses and the quantitation of virus 

infectivity by FA.

3.2 Preparation of equipment/instrumentation

3.2.1 On the day of inoculation, set a water bath at 36°± 2°C.

3.2.2 On the day of the FA, prepare a humidity chamber in the aerobic incubator 

by filling a pan in the bottom with DI.

3.3 Preparation of reagents/control procedures

3.3.1 Pretreated Lab-Tek®Slides

Two days before the day of inoculation, prepare sufficient Lab-Tek®Slides to 

allow 25 chambers for controls and 20 chambers for each Test Vaccine. Using a 

2-mL self-refilling repetitive syringe, add 0.4 mL/chamber of Growth Medium to 

all chambers of the Lab-Tek®Slides; incubate at 37°± 2°C for 48 ± 8 hours. 

3.3.2 Preparation of DKp Slides

Cells are prepared from healthy, confluent DKp cell cultures at second or third 

passage. On the day of test initiation, decant Growth Medium from slides and add 

0.4 mL/chamber of approximately 104.9 to 105.2 cells/mL suspended in Growth 

Medium into all chambers of the Lab-Tek®Slides. Incubate at 37°± 2°C in a CO2 

incubator and use within 4 hours.

3.3.3 Preparation of CDV Positive Control

1. On the day of inoculation, rapidly thaw a vial of CDV Positive Control

in a 36°± 2°C water bath.

2. Dispense 1.8 mL MEM into sufficient 12 x 75-mm polystyrene tubes 

to bracket the expected endpoint according to the CVB Reagent Data 

Sheet and label appropriately (e.g., 5 tubes, labeled 10-1through 10-5, 

respectively).

3. With a 200-μL pipettor, transfer 200 μL of the CDV Positive Control

to the tube labeled 10-1; mix by vortexing.
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4. Using a new pipette tip, transfer 200 μL from the 10-1labeled tube 

(Step 3) to the 10-2 tube; mix by vortexing.

5. Repeat Step 4 for each of the subsequent dilutions, transferring 200 

μL from the previous dilution to the next dilution tube. 

3.3.4 Preparation of working antisera and/or FA conjugate solutions

For FA staining, dilute the Anti-CDV Conjugate; for IFA staining, dilute CDV 

antiserum and Anti-dog conjugate. Reagents are diluted in DPBS according to the 

manufacturer's recommendations.

3.4 Preparation of the sample

3.4.1 The initial test of a Test Vaccine will be with a single vial (a single sample 

from 1 vial). On the day of inoculation, using a sterile 1.0-mL syringe and an 

18-gauge x 1 1/2-inch needle, rehydrate a vial of the Test Vaccine by transferring 

1.0 mL for 1-mL-dose vaccines, 0.5 mL for 1/2-mL-dose vaccines, etc., of the 

provided diluent into the vial containing the lyophilized Test Vaccine; mix by 

vortexing. Incubate for 15 ± 5 minutes at room temperature.

3.4.2 For multifraction CDV vaccines, neutralize the non-CDV fractions with 

antiserum specific to each virus fraction.

1. Prepare dilutions of each neutralizing non-CDV antiserum according 

to the CVB Reagent Data Sheet or the manufacturer’s instructions.

2. Dispense 200 μL of each of the required neutralizing antisera into a 12 

x 75-mm polystyrene tube, labeled 10-1, and q.s. to 1.8 mL with MEM. 

For example, to neutralize the 3 non-CDV viral fractions of a 

CDV/CAV/CPI/CPV vaccine, dispense 200 μL of each of the diluted 

CAV, CPI, and CPV antisera into the tube labeled 10-1; add 1.2 mL of 

MEM to obtain a final volume of 1.8 mL. 

3. Pipette 200 μL of the reconstituted Test Vaccine to the labeled tube to 

yield a 10-1dilution; mix by vortexing.

4. Incubate at room temperature for 30 ± 5 minutes. 

3.4.3 For Test Vaccines containing only the CDV fraction, the 10-1dilution is 

prepared by adding 200 μL of the Test Vaccine to 1.8 mL of MEM in a 12 x 

75-mm polystyrene tube, labeled 10-1; mix by vortexing. 
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3.4.4 Vaccine tenfold dilutions

1. Dispense 1.8 mL MEM into each of 3, 12 x 75-mm polystyrene tubes, 

labeled 10-2through 10-4(or more if the expected CDV endpoint of the 

Test Vaccine is higher than 10-4). 

2. Using a new pipette tip, transfer 200 μL from the tube labeled 10-1to 

the next dilution tube; mix by vortexing.

3. Repeat Step 2 to the remaining tubes, transferring 200 μL from the 

previous dilution to the next dilution tube, until the tenfold dilution series 

is completed.

4. Performance of the Test

4.1 Label the DKp Slides and inoculate each of 5 chambers/dilution with 100 μL of 

the Test Vaccine. In a similar manner, inoculate 5 chambers/dilution of the CDV Positive 

Control (with dilutions 10-5through 10-2from Section 3.3.3). Change tips between each 

unique sample (i.e., each Test Vaccine and the CDV Positive Control), but tip changes 

are not necessary between each dilution in a series if pipetting from the most dilute to the 

most concentrated within that series (e.g., 10-5through 10-2).

4.2 Five uninoculated chambers serve as negative cell controls.

4.3 Incubate slides in a 36°± 2°C CO2 incubator for 5 days ± 1 day.

4.4 After incubation, decant the media from the DKp Slides into an autoclavable 

waste container. Remove the plastic chambers by tipping them away from the slide, 

leaving the rubber gasket attached to the slide.

4.5 Place the slides in a rack, set in a glass staining dish filled with DPBS, and 

incubate 15 ± 5 minutes at room temperature.

4.6 Place the rack in a glass staining dish filled with 100% acetone for 15 ± 5 minutes

at room temperature. Remove and allow to air dry.

4.7 For FA: pipette 75 ± 25 μL of the Working Anti-CDV Conjugate into each 

chamber of the DKp Slides; for IFA: pipette 75 ± 25 μL of the Working CDV antiserum 

into each chamber of the DKp Slides; incubate at 36°± 2°C in the high humidity, aerobic 

incubator for 30 ± 5 minutes.

4.8 Remove the DKp Slides from the incubator, decant and wash per Section 4.5.
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4.9 For IFA: pipette 75 ± 25 μL of the Working Anti-Dog conjugate into each 

chamber of the DKp Slides; incubate at 36°± 2°C in the high humidity, aerobic incubator 

for 30 ± 5 minutes. Wash per Section 4.5.

4.10 Rinse by quickly dipping the rack of slides in a glass staining dish filled with DI; 

allow to air dry. 

4.11 Read at 100X or 200X magnification on a UV-light microscope and examine cells 

for typical apple-green cytoplasmic fluorescence.

4.11.1 Chambers containing 1 or more cells displaying specific fluorescence are 

considered to be positive for CDV virus. 

4.11.2 Record results as the number of FA-positive chambers versus the total 

number of chambers examined for each dilution of a Test Vaccine and the CDV 

Positive Control.

Calculate the CDV endpoints of the Test Vaccine and the CDV Positive Control 

using the Spearman-Kärber method as modified by Finney. The titers are 

expressed as log10 50% fluorescent antibody infective dose (FAID50). 

Example:

10-1dilution of Test Vaccine = 5/5 chambers FA positive

10-2dilution of Test Vaccine = 4/5 chambers FA positive

10-3 dilution of Test Vaccine = 2/5 chambers FA positive

10-4dilution of Test Vaccine = 0/5 chambers FA positive

Spearman-Kärber formula:

Test Vaccine titer = (X - d/2 + [d • S]) where:

X = log10 of highest dilution with all chambers FA positive (1)

d = log10 of tenfold dilution factor (1)

S = sum of proportions of chambers FA positive for all dilutions tested:

5 4 2 0 11

5 + 5 + 5 + 5 = 5 = 2.2

Test Vaccine titer = (1 - 1/2) + (1 • 2.2) = 2.7
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Adjust the titer to the recommended Test Vaccine dose as follows:

A. divide the Test Vaccine Dose by the Inoculation Dose where:

Test Vaccine Dose = manufacturer’s recommended vaccination dose (for this 

Test CDV vaccine, the recommended dose is 1 mL)

Inoculation Dose = amount of diluted Test vaccine added to each chamber of the 

Lab-Tek slide (for this test CDV vaccine, the inoculation dose is 0.1 mL)

 ___1 mL dose___

 0.1 mL inoculum = 10

B. calculate log10 of value in A and add it to the Test Vaccine titer as illustrated 

below:

 Log of 10 = 1.0

Test Vaccine titer = 2.7 + 1.0 = 3.7.

Therefore, the titer of the CDV Test Vaccine is 103.7 FAID50/mL.

5. Interpretation of the Test Results - Valid Assay

5.1 The calculated titer of the CDV Positive Control must fall within plus or minus 2 

standard deviations (± 2 SD) of its mean titer, as established from a minimum of 10 

previously determined titers.

5.2 The lowest inoculated dilution of the CDV Positive Control must exhibit a 100% 

positive FA/IFA reaction (5/5). If an endpoint is not reached (1 or more chambers are 

FA/IFA positive at the highest dilution), the titer is expressed as “greater than or equal 

to” the calculated titer. If an endpoint is critical to testing, the highest (most dilute) must 

exhibit no positive FA/IFA reaction (0/5).

5.3 The uninoculated cell controls must not exhibit any cytopathic effect (CPE), 

specific CDV virus fluorescence, or cloudy media that would indicate contamination.

5.4 If the validity requirements are not met, then the assay is considered a NO TEST

and can be retested without prejudice.

5.5 If the validity requirements are met and the titer of the Test Vaccine is greater 

than or equal to the titer contained in the Animal and Plant Health Inspection Service 

filed Outline of Production for the product under test, the Test Vaccine is considered 

SATISFACTORY.
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5.6 If the validity requirements are met but the titer of the Test Vaccine is lower than 

the required minimum, the Test Vaccine is retested in accordance with 9 CFR, Part 113.8.

6. Report of Test Results

Results are reported as FAID50 per dose of Test Vaccine.

7. References

7.1 Title 9, Code of Federal Regulations, part 113.306, U.S. Government Printing

Office, Washington, DC.

7.2 Cottral, GE (Ed.) Manual of standardized methods for veterinary microbiology. 

Comstock Publishing Associates, Ithaca and London, 1978, pg. 731.

7.3 Finney, DJ Statistical methods in biological assay. Griffin, London. 3rd ed., 

1978, pp. 394-401.

8. Summary of Revisions

Version .07

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

to keep the same next review date for the document.

Version .06

 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 .05

 The Contact information has been updated.

 CDV Positive Control, Rockborn strain is no longer available from the CVB and all 

references to it have been removed from the document.

Version .04

 The term “Reference” has been changed to “Positive Control” throughout the 

document.
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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:

 2.2.5.2 The amount of sodium bicarbonate (NaHCO3) has been changed from 2.2 g 

to 1.1 g.

 2.2.5.7 Penicillin and streptomycin have been deleted.

 4.11.2 Additional steps have been added to clarify the titer calculations by the 

Spearman-Kärber formula.

 5.1.2 Recording the rate of the FA positive reaction for validity requirements.

 “Reference and Reagent Sheet” has been changed to “Reagent Data Sheet” 

throughout the document.

 The refrigeration temperatures have been changed from 4° ± 2°C to 2°- 7°C. This 

reflects the parameters established and monitored by the Rees system.

 “Test Serial” has been changed to “Test Vaccine” throughout the document.

 The footnotes have been deleted with any pertinent references now noted next to the 

individual items.