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SAM 304.05

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United States Department of Agriculture

Center for Veterinary Biologics

Testing Protocol

SAM 304

Supplemental Assay Method for Titration of Infectious Canine Hepatitis 

Virus in Primary Canine Kidney Cell Culture

Date: October 30, 2014

Number: SAM 304.05

Supersedes: SAM 304.04, 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: 05Jan15

Geetha B. Srinivas, Section Leader

Virology

/s/Byron E. Rippke Date: 09Jan15

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 Infectious Canine Hepatitis Virus 

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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 Test Vaccine

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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1. Introduction

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

modified-live infectious canine hepatitis (ICH) or canine adenovirus (CAV) type 1 virus 

vaccines for viral content. ICH endpoint is determined by cytopathic effect (CPE) in 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 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 light (Model CK, Olympus America Inc.)

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

2.1.5 Syringe, 2-mL self-refilling, repetitive

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 ICH Positive Control, Mirandola strain of ICH virus, type 1

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

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 Primary dog kidney (DKp) cell culture, 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, then 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)

c. 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 the solution in Step 5 with 

constant mixing.

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

and add slowly to the solution in 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.

2.2.7 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 a water bath at 36°± 2°C.
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3.3 Preparation of reagents/control procedures

3.3.1 Preparation of DKp cell culture plates (Test Plates) 

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

passage. On the day of test initiation, using the 12-channel micropipettor, add 

200 μL/well of approximately 104.7 to 105.2 cells/mL suspended in Growth 

Medium into all wells of a 96-well cell culture plate. Prepare 1 Test Plate for the 

controls and 1 Test Vaccine. Each additional Test 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 ICH Virus Positive Control

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

Control in a water bath. 

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

bracket the expected endpoint according to the CVB Reagent Data Sheet, 

and label (e.g., 8 tubes labeled 10-1through 10-8, respectively).

3. With a 200-μL pipettor, transfer 200 μL of the ICH Virus Positive 

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

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

3.3.3 Prepare dilutions of each neutralizing non-ICH antiserum according to the 

CVB Reagent Data Sheet or manufacturer’s instructions.

3.4 Preparation of the Test Vaccine

3.4.1 Conduct the initial test of a Test Vaccine 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 1.0-mL-dose vaccine, 0.5 mL for a 

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

mix by vortexing. Incubate for 15 ± 5 minutes at room temperature.
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3.4.2 For multifraction ICH vaccines, neutralize the non-ICH fractions with 

antiserum specific to each virus fraction. 

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

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

For example, to neutralize the 3 viral non-ICH Virus components of a 

CDV/ICH/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.

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

yield a 10-1dilution; mix by vortexing. 

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

3.4.3 For vaccines containing only the ICH 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. 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; appropriately label (e.g., 8 tubes, labeled 10-1through 10-8, 

respectively). 

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 On the day of test initiation, label the Test 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 of the ICH Virus Positive Control 

(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 ICH Virus 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).
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4.2 Eight uninoculated wells serve as negative cell controls.

4.3 Incubate the inoculated Test Plates in the CO2 incubator for 11 days ± 1 day.

4.4 After incubation, read at 100X or 200X magnification on an inverted light 

microscope and examine cells for CPE characterized by cell rounding and lysis.

4.4.1 Wells displaying one or more CPE foci are considered to be positive for 

ICH Virus.

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 ICH Virus Positive

Control.

4.5 Calculate the ICH endpoints of the Test Vaccine and the ICH Virus Positive

Control using the Spearman-Kärber method as commonly 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

10-4dilution 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 proportion 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 Test Vaccine dose as follows:

A. divide the Test Vaccine Dose by the Inoculation Dose

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

ICH test vaccine, the recommended dose is 2 mL) 
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Inoculation Dose = amount of diluted Test Vaccine added to each well of the 

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

 2 mL dose = 80

0.025 mL inoculum

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

below:

Log of 80 = 1.9

Test Vaccine titer = 3.25 + 1.9 = 5.15

Therefore the titer of the ICH Test Vaccine is 105.15 TCID50/2mL.

5. Interpretation of the Test Results

5.1 Valid assay

5.1.1 The calculated titer of the ICH Virus 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 ICH Virus Positive Control must 

exhibit a 100% positive CPE reaction (8/8). If an endpoint is not reached (1 or 

more wells are CPE positive wells 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 CPE reaction (0/8).

5.1.3 The uninoculated cell controls 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.

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 lower than the titer contained in 

the APHIS filed Outline of Production for the product under test, the Test Vaccine may 

be retested in accordance with 9 CFR, Part 113.8.b. 
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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, 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.

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.
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 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.

 “Reference and Reagent 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.