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SAM 317.05

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United States Department of Agriculture

Center for Veterinary Biologics

Testing Protocol

SAM 317

Supplemental Assay Method for Titration of Canine Adenovirus in Canine 

Kidney Cell Culture

Date: November 13, 2014

Number: SAM 317.05

Supersedes: SAM 317.04, June 30, 2011

Standard Requirement: 9 CFR 113.305

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

Peg A. Patterson

Approvals: /s/Geetha B. Srinivas Date: 03Dec14

Geetha B. Srinivas, Section Leader

Virology

/s/Byron E. Rippke Date: 10Dec14

Byron E. Rippke, Director

Policy, Evaluation, and Licensing

Center for Veterinary Biologics

/s/Rebecca L.W. Hyde Date: 15Dec14

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 Adenovirus in Canine Kidney 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

6. Report of Test Results

7. References

8. Summary of Revisions
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Supplemental Assay Method for Titration of Canine Adenovirus in Canine Kidney Cell Culture

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1. Introduction

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

modified-live canine adenovirus (CAV) vaccines for viral content. CAV endpoint is determined 

by viral cytopathic effect (CPE) in a Madin-Darby canine kidney (MDCK) cell line.

2. Materials

2.1 Equipment/instrumentation

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

below.

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

Scientific Inc.)

2.1.2 Water bath, 36°± 2°C 

2.1.3 Microscope, inverted bright light (Model CK, Olympus America Inc.)

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

2.1.5 Syringe, self-refilling, repetitive, 2-mL

2.1.6 Pipettor with tips and/or motorized microliter pipette 

2.1.7 Micropipettor, 300-μL x 12-channel

2.1.8 Pipette-aid

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 CAV Positive Control: Mirandola strain of CAV Type 1 or Manhattan 

strain of CAV Type 2

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

fractions present in multifraction vaccines, e.g. canine parainfluenza virus (CPI), 

canine parvovirus (CPV), canine distemper virus (CDV), etc.
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2.2.3 MDCK cell line, free of extraneous agents as tested by title 9, Code of 

Federal Regulations (9 CFR) 

2.2.4 Minimum essential medium (MEM)

1. 9.61 g MEM with Earles salts without bicarbonate

2. 1.1 g sodium bicarbonate (NaHCO3) 

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, and add to the solution in Step 3 with constant 

mixing.

5. Q.S. to 1000 mL with DI and 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.

2.2.5 Growth Medium

1. 940 mL MEM

2. Aseptically add:

a. 50 mL gamma-irradiated fetal bovine serum (FBS) 

b. 10 mL L-glutamine (200 mM)

3. Store at 2°- 7°C.

2.2.6 Dulbecco’s phosphate buffered saline (DPBS)

1. 8.0 g sodium chloride (NaCl) 

2. 0.2 g potassium chloride (KCl)

3. 0.2 g potassium phosphate, monobasic, anhydrous (KH2PO4)
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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. 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 5 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.

2.2.7 Cell culture plates, 96-well

2.2.8 Polystyrene tubes, 12 x 75-mm

2.2.9 Pipettes, 10-mL

2.2.10 Reagent reservoir

2.2.11 Syringe, 1-mL tuberculin 

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

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 CPE.

3.2 Preparation of equipment/instrumentation

On the day of test initiation, set the water bath at 36°± 2°C.
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3.3 Preparation of reagents/control procedures

3.3.1 Preparation of MDCK cell culture plates (MDCK Plates)

Cells are prepared from healthy, confluent MDCK cells. On the day of test 

initiation, using a 12-channel micropipettor, add 200 μL/well of 104.7 to 105.2

cells/mL suspended in Growth Medium into all wells of a 96-well cell culture 

plate. Prepare 1 MDCK Plate for the controls and the first Test Vaccine. Each 

additional MDCK Plate allows testing of 3 additional Test Vaccines. Incubate at 

36°± 2°C in a CO2 incubator and use within 4 hours. 

3.3.2 Preparation of CAV Positive Control

1. On the day of test initiation, rapidly thaw a vial of CAV Positive 

Control in the 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 (for example: 8 tubes labeled 10-1through 10-8, 

respectively).

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

to the first tube labeled 10-1; mix by vortexing.

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 until the tenfold 

dilution series is completed.

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 with the 

provided diluent by transferring 1.0 mL for a 1-mL-dose vaccine, 0.5 mL for 

1/2-mL-dose vaccines, etc., into the vial containing the lyophilized Test Vaccine; 

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

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

antiserum specific to each virus fraction. 
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1. Prepare a dilution of each neutralizing non-CAV antiserum in DPBS 

according to the CVB Reagent Data Sheet or as determined for that 

specific antiserum.

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

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

For example, to neutralize 3 non-CAV viral components of a 

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

CDV, 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 vaccines containing only the CAV 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. 

3.4.4 Serial tenfold dilutions

1. Using a 2-mL self-refilling repetitive syringe, dispense 1.8 mL MEM 

into each of 4, 12 x 75-mm polystyrene tubes labeled 10-2through 10-5(or 

more if the expected CAV endpoint of the Test Vaccine is higher than 

10-5). 

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

the next dilution tube labeled 10-2; 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 MDCK Plates and inoculate each of 8 wells/dilution with 25 μL of the 

Test Vaccine, starting with the highest dilution (most dilute). In a similar manner, 

inoculate 8 wells/dilution of the CAV Positive Control (with dilutions 10-8through 10-5

for the example in Section 3.3.2(2)). Change tips between each unique sample (i.e., each 

Test Vaccine and the CAV 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., 10-8through 10-5). This becomes the Test Plate. Additional Test Vaccines 
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may be inoculated onto other MDCK Plates in a similar manner, 3 Test Vaccines per Test 

Plate.

4.2 Eight uninoculated wells on the initial Test Plate serve as a Negative Cell Control.

4.3 Incubate the Test Plates in a 36°± 2°C CO2 incubator for 11 days ± 1 day.

4.4 After incubation, read the Test Plate at 100X or 200X magnification on an 

inverted light microscope and examine cells for CPE. CAV CPE is characterized by cell 

rounding and lysis.

4.4.1 Wells displaying 1 or more areas of CPE are considered to be positive for 

CAV.

4.4.2 Record results as the number of CPE positive wells versus total number of 

wells examined for each dilution of the Test Vaccine and the CAV Positive

Control.

4.5 Calculate the CAV endpoints of the Test Vaccine and the CAV Positive Control 

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

log10, 50% tissue culture infective doses (TCID50).

Example:

10-2dilution of Test Vaccine = 8/8 wells CPE positive

10-3dilution of Test Vaccine = 5/8 wells CPE positive

10-4 dilution of Test Vaccine = 1/8 wells CPE positive

10-5dilution of Test Vaccine = 0/8 wells CPE positive

Spearman-Kärber formula:

Test Vaccine Titer = (x – d/2 + [d ● S]), where:

X = log10 of highest dilution with all wells CPE positive (2)

d = log10 of tenfold dilution factor (1)

S = sum of proportions of wells CPE positive for all dilutions tested:

8 5 1 0 14

8 + 8 + 8 + 8 = 8 = 1.75

Test Vaccine titer = (2 – 1/2) + (1 ● 1.75) = 3.25

Adjust the titer to the recommended Test Vaccine dose as follows:
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A. divide the Test Vaccine Dose by the Inoculation Dose

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

CAV vaccine, the recommended dose is 1 mL)

Inoculation Dose = amount of diluted Test Vaccine added to each well of the 

Test Plate (for this test CAV vaccine, the inoculation dose is 0.025 mL)

1 mL dose = 40

0.025 mL

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

below:

Log of 40 = 1.6

Test Vaccine Titer = 3.25 + 1.6 = 4.85

Therefore the titer of the CAV Test Vaccine is 104.85 TCID50/mL.

5. Interpretation of the Test Results

5.1 Valid assay

5.1.1 The calculated titer of the CAV 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.

5.1.2 The lowest inoculated dilution of the CAV Positive Control must induce 

CPE in 100% of the wells (8/8). If an endpoint is not reached (1 or more wells are 

CPE 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 CPE (0/8).

5.1.3 The Uninoculated Cell Control must not exhibit any CPE, degradation, or 

cloudy media that would indicate contamination.

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

and can be retested without prejudice.
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5.3 In a valid test, if the titer of the Test Vaccine is greater than or equal to the titer 

contained in the Animal and Plant Health Inspection Service (APHIS) filed Outline of 

Production for the product under test, the Test Vaccine is considered SATISFACTORY.

5.4 In a valid test, if the titer of the Test Vaccine is less than the required minimum 

contained in an APHIS filed Outline of Production for the product under test, the Test 

Vaccine is retested in accordance with 9 CFR, Part 113.8.

6. Report of Test Results

Results are reported as TCID50 per dose of Test Vaccine.

7. References

7.1 Title 9, Code of Federal Regulations, part 113.305, 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, 1978. Statistical method in biological assay. Griffin, London. 3rd ed., 

1978, pp. 394-401.

8. Summary of Revisions

Version .05

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

to keep the same next review date for the document.

Version .04

 The Contact information has been updated.

Version .03

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

document.
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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: 

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

to 1.1 g.

 2.2.4.7 Penicillin and streptomycin have been deleted.

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

Spearman-Kärber formula.

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

 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.

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

throughout the document.

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

individual items.