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SAM 309.05

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United States Department of Agriculture

Center for Veterinary Biologics

Testing Protocol

SAM 309

Supplemental Assay Method for Titration of Canine Parainfluenza Virus in 

Vero Cell Culture

Date: October 30, 2014

Number: SAM 309.05

Supersedes: SAM 309.04, June 30, 2011

Standard Requirement: 9 CFR, Part 113.316

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

Peg A. Patterson

Approvals: /s/Geetha B. Srinivas Date: 09Jan15

Geetha B. Srinivas, Section Leader

Virology

/s/Byron E. Rippke Date: 13Jan15

Byron E. Rippke, Director

Policy, Evaluation, and Licensing

Center for Veterinary Biologics

/s/Rebecca L.W. Hyde Date: 13Jan15

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 Parainfluenza Virus in Vero 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 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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Supplemental Assay Method for Titration of Canine Parainfluenza Virus in Vero Cell Culture

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1. Introduction

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

modified-live canine parainfluenza (CPI) virus vaccines for viral content. Presence or absence of 

CPI virus is determined by hemadsorption (HAd) of guinea pig red blood cells (GPRBC).

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

2.1.3 Centrifuge and rotor (Beckman Coulter)

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

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

2.1.6 Syringe, self-refilling repetitive, 2-mL

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

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 CPI Positive Control Virus, D008 strain

2.2.2 Monospecific antisera, free of CPI antibody, which neutralize the non-CPI 

virus fractions present in multifraction vaccines (e.g., canine distemper virus 

(CDV) and canine adenovirus (CAV))
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Supplemental Assay Method for Titration of Canine Parainfluenza Virus in Vero Cell Culture

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2.2.3 African green monkey kidney (Vero) 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 reagents in Steps 1 and 2 with 900 mL deionized water (DI).

4. Add 5.0 g lactalbumin hydrolysate or edamine to 10 mL DI, and heat 

to 60°± 2°C until dissolved. 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 Alsevers Solution

1. 8.0 g sodium citrate (C6H5Na3O7•2H2O)

2. 0.55 g citric acid (C6H8O7•H2O)
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3. 4.2 g sodium chloride (NaCl)

4. 20.5 g glucose (C6H12O6)

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

6. Sterilize through a 0.22-μm filter.

7. Store at 2°- 7°C.

2.2.7 Dulbecco’s phosphate buffered saline free of calcium (Ca++) and 

magnesium (Mg++) ions.

1. 8.0 g NaCl

2. 0.2 g potassium chloride (KCl)

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

 

4. Dissolve reagents in Steps 1 through 3 with 900 mL DI.

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

mL DI, and heat to 60°± 2°C until dissolved. Add to Step 4 with constant 

mixing.

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

7. Sterilize through a 0.22-μm filter.

8. Store at 2°- 7°C.

2.2.8 Dulbecco’s phosphate buffered saline (DPBS)

1. 8.0 g NaCl

2. 0.2 g KCl

3. 0.2 g KH2PO4

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

5. Dissolve reagents in Steps 1 through 4 with 900 mL DI.
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6. Add 1.03 g Na2HPO4 to 10 mL DI, and 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.

2.2.9 Guinea pig blood in an equal volume of Alsevers Solution

2.2.10 Cell culture plates, 24-well

2.2.11 Polystyrene tubes, 12 x 75-mm

2.2.12 Pipettes, 10-mL and 25-mL

2.2.13 Conical tubes, 50-mL

2.2.14 Syringe, 1-mL tuberculin, and needle, 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 HAd.

3.2 Preparation of equipment/instrumentation

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

3.3 Preparation of reagents/control procedures

3.3.1 Preparation of Vero cell culture plates (Test Plates)

Cells are prepared from healthy, confluent Vero cells that are maintained by 

passing every 3 to 4 days. One day prior to test initiation, add 1.0 mL/well of 104.7

to 105.2 cells/mL cells suspended in Growth Medium into all wells of a 24-well 

cell culture plate. Prepare 1 Vero plate for the controls and 1 for each Test 
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Vaccine. These become the Test Plates. Incubate at 36°± 2°C in a CO2 incubator 

for one day.

3.3.2 Preparation of CPI Positive Control Virus

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

Control Virus in a 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; appropriately label (e.g., 9 tubes, labeled 10-1through 10-9, 

respectively).

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

Virus 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 of the previous dilution to the next dilution tube until the tenfold 

dilution series is completed. 

3.3.3 Prepare a dilution of each neutralizing non-CPI antiserum in DPBS 

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

specific antiserum. 

3.3.4 Preparation of washed guinea pig red blood cells (GPRBC)

1. Transfer 20 mL of the guinea pig blood into a 50-mL conical tube.

 

2. Q.S. to 50 mL with Alsevers Solution, and mix by inverting several 

times.

 

3. Centrifuge for 15 ± 5 minutes at 400 x g (1500 rpm in a Beckman J6B 

centrifuge with JS-4.0 rotor) at 4°± 2°C.

 

4. Remove supernatant and buffy coat by pipetting.

5. Repeat steps in Steps 2 through 4 for a total of 3 washes.
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6. Add an equal volume of Alsevers Solution to the washed GPRBC. 

Store at 2°- 7°C; use within 1 week of collection.

 

3.3.5 0.5% GPRBC Suspension for the HAd assay

1. On the day of conducting the HAd test, pipette 500 μL of washed, 

packed GPRBC into 100 mL of Ca++, Mg++-Free DPBS, rinsing the pipette 

thoroughly to remove the GPRBC.

2. Mix by inverting, store at 2°- 7°C, and use within 1 week of collection.

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 1-mL-dose vaccines, 0.5 mL for 

0.5-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 CPI vaccines, neutralize the non-CPI viral fractions with 

antiserum specific to each non-CPI virus fraction. It is not necessary to neutralize 

canine parvovirus (CPV) since CPV is not expected to replicate in confluent Vero 

cells. 

1. Prepare dilutions of each neutralizing non-CPI antiserum according to 

the CVB Reagent Data Sheet or manufacturer’s instructions. 

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

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

For example, to neutralize 2 non-CPI viral fractions in a 3-fraction 

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

CAV antiserums into the tube labeled 10-1; add 1.4 mL of MEM to obtain 

a final volume of 1.8 mL.

 

3. Pipette 200 μL of the reconstituted Test Vaccine to the 10-1labeled 

tube containing the neutralizing antisera; mix by vortexing. 

4. Incubate at room temperature for 60 ± 10 minutes.

3.4.3 For Test Vaccines containing only the CPI viral fraction, prepare the 10-1

dilution by mixing 200 μL of the reconstituted Test Vaccine with 1.8 mL of MEM 

in a labeled 12 x 75-mm polystyrene tube. 
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3.4.4 Serial tenfold dilutions

1. Dispense 1.8 mL MEM into each of 5, 12 x 75-mm polystyrene tubes 

labeled 10-2through 10-6(or more if the expected CPI endpoint of the Test 

Vaccine is higher than 10-6). 

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 On the day of test inoculation, label the Test Plates and inoculate each of 5 

wells/dilution with 200 μL of dilutions 10-6through 10-3of each Test Vaccine. In a 

similar manner, identify and inoculate 5 wells/dilution of the CPI Positive Control Virus 

(with dilutions 10-9through 10-6for the example in Section 3.3.2). Change tips between 

each unique sample (i.e., each Test Vaccine and the CPI Positive Control Virus); tip 

changes are not necessary between dilutions in a series if pipetting from the most dilute 

to the most concentrated within that series (e.g., 10-9through 10-6).

4.2 Five uninoculated wells serve as Negative Cell Controls.

4.3 Incubate the Test Plates in a 36°± 2°C CO2 incubator for 8 days ± 1 day.

4.4 After incubation, perform the HAd assay on the cell monolayers with a 0.5% 

GPRBC suspension.

4.4.1 Decant the media from the plates into an autoclavable container.

4.4.2 Submerge each Test Plate in a reservoir of Ca++, Mg++-Free DPBS and 

decant.

4.4.3 Mix the 0.5% GPRBC suspension by gentle inversion, and add 

1.0 mL/well.

4.4.4 Incubate at 2°- 7°C for 25 ± 5 minutes.

4.4.5 Wash Test Plates by repeating Section 4.4.2 twice. 
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4.4.6 Read the wet Test Plate at 100X magnification on an inverted light 

microscope, and examine CPI-infected vero cells for HAd.

1. Wells displaying GPRBC adsorbed onto the cell monolayer are 

considered to be positive for CPI. 

2. Record the results as the number of HAd- positive wells versus total 

number of wells examined for each dilution of the Test Vaccine and the 

CPI Positive Control Virus.

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

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

expressed as log10 50% HAd infective doses (HAdID50).

Example:

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

10-4dilution of Test Vaccine = 4/5 wells HAd positive

10-5 dilution of Test Vaccine = 1/5 wells HAd positive

10-6dilution of Test Vaccine = 0/5 wells HAd positive

Spearman-Kärber formula:

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

X = log10 of highest dilution with all wells HAd positive (3)

d = log10 of tenfold dilution factor (1)

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

5 4 1 0 10

5 + 5 + 5 + 5 = 5 = 2.0

CPI Test Vaccine titer = (3 - 1/2) + (1 • 2.0) = 4.5

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 CPI vaccine, the recommended dose is 1 mL)
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Inoculation Dose = amount of diluted Test vaccine added to each well of 

the Test Plate (for this test CPI vaccine, the inoculation dose is 0.2 mL)

 ___1 mL dose 

 0.2 mL inoculum = 5

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

below:

 Log of 5 = 0.7

CPI Test Vaccine titer = 4.5 + 0.7 = 5.2

 

Therefore, the titer of the CPI Test Vaccine is 105.2 HAdID50/mL.

5. Interpretation of the Test Results

5.1 Validity requirements

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

exhibit a 100% positive HAd reaction (5/5). If an endpoint is not reached (1 or 

more wells are HAd 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 HAd reaction (0/5).

5.1.3 The Negative Cell Controls shall not exhibit degradation, HAd, or cloudy 

media indicative of contamination.

5.2 If the validity requirements are not met, 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 less 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 113.8.
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6. Report of Test Results

Results are reported as HAdID50 per dose of Test Vaccine. 

7. References

7.1 Title 9, Code of Federal Regulations, part 113.316, U.S. Government Printing 

Office, Washington, DC.

7.2 Cottral, GE, (Ed.), 1978. Manual of standardized methods for veterinary 

microbiology. Comstock Publishing Associates, Ithaca, pg. 731.

7.3 Finney, DJ, 1978. Statistical method in biological assay. Griffin, London. 3rd 

edition, 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 the 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.