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SAM 316.05

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United States Department of Agriculture

Center for Veterinary Biologics

Testing Protocol

SAM 316

Supplemental Assay Method for the Titration of Canine Parvovirus in Cell 

Culture

Date: November 14, 2014

Number: SAM 316.05

Supersedes: SAM 316.04, June 30, 2011

Standard Requirement: 9 CFR 113.317

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

Peg A. Patterson

Approvals: /s/Geetha B. Srinivas Date: 23Dec14

Geetha B. Srinivas, Section Leader

Virology

/s/Byron E. Rippke Date: 29Dec14

Byron E. Rippke, Director

Policy, Evaluation, and Licensing

Center for Veterinary Biologics

/s/Rebecca L.W. Hyde Date: 29Dec14

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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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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1. Introduction

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

modified-live canine parvovirus (CPV) vaccines for viral content. The method uses the Crandell 

feline kidney (CRFK) cell line as the test system. CPV endpoint is determined by staining 

inoculated cell cultures by an indirect fluorescent antibody (IFA) staining method.

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 Incubator aerobic (Model 2, Precision Scientific)

2.1.3 Water bath

2.1.4 Microscope, ultraviolet (UV) light (Model BH2, Olympus America)

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

2.1.6 Micropipettor, tips, and/or motorized microliter pipette or equivalent and 

tips

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

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 CPV Positive Control, KB5 strain

2.2.2 CRFK cell culture, free of extraneous agents as tested by title 9, Code of

Federal Regulations (9 CFR)

2.2.3 Feline panleukopenia virus antiserum (FPV Antiserum)or Canine 

Parvovirus antiserum (CPV Antiserum)
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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. Add to 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.

2.2.5 Growth Medium

1. 920 mL of MEM

2. Aseptically add:

a. 70 mL gamma-irradiated fetal bovine serum (FBS)

b. 10 mL L-glutamine (200 mM)

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)

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

5. Dissolve reagents with 900 mL DI.
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6. Dissolve 1.03 g sodium phosphate, dibasic, anhydrous (Na2HPO4) 

with 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; 

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.

2.2.7 Cell culture glass slides, 8-well (Lab-Tek®Slides)

2.2.8 Polystyrene tubes, 12 x 75-mm 

2.2.9 Appropriate anti-species IgG (H&L) fluorescein isothiocyanate labeled 

conjugate (Anti-species Conjugate)

2.2.10 100% acetone

2.2.11 Syringe, 1-mL and needle, 20-gauge x 1 1/2-inch

2.2.12 Pipette-Aid

2.2.13 Disposal transfer pipette, 4.6-mL

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 of animal viruses and the quantitation of virus infectivity by the IFA

staining method.

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 IFA test, prepare a humidity chamber in the aerobic 

incubator by filling a pan in the bottom with DI.
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3.3 Preparation of reagents/control procedures

3.3.1 Preparation of CRFK Slides

1. Cells are prepared from healthy, confluent CRFK cells, that are 

maintained by passing every 3 to 4 days. On the day of test initiation, add 

0.4 mL/well of 105.2 to 105.4 cells/mL diluted in Growth Media into all 

wells of a Lab-Tek®slide. Prepare sufficient Lab-Tek®Slides to allow 25 

wells for controls and 20 wells for each Test Vaccine. Incubate at 36°± 

2°C in a CO2 incubator. These become the CRFK Slides.

2. Use seeded CRFK Slides within 4 hours.

3.3.2 Preparation of the CPV Working Positive Control

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

in a 36°± 2°C water bath.

2. Dispense 1.8 mL of MEM into each of 7, 12 x 75-mm polystyrene 

tubes labeled 10-1through 10-7.

3. Transfer 200 μL of the CPV Positive Control to the tube labeled 10-1;

discard pipette tip. Mix by vortexing.

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

(Step 3) to the 10-2tube; discard pipette tip. Mix by vortexing.

5. Repeat Step 4 for each subsequent dilution, transferring 200 μL from 

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

series is completed.

3.3.3 Preparation of Working FPV or CPV Antiserum

On the day of the IFA test, dilute FPV or CPV Antiserum in DPBS to the IFA 

working dilution on the CVB Reagent Data Sheet or as determined for that 

specific antiserum. 

3.3.4 Preparation of Working Anti-species Conjugate

On the day of the IFA test, dilute Anti-species Conjugate in DPBS to the working 

dilution according to the manufacturer’s recommendations.
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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, rehydrate a vial of the Test Vaccine by 

transferring 1.0 mL for a 1-mL-dose vaccine, 0.5 mL for 1/2-mL-dose vaccines, 

etc., of the provided diluent into the vial containing the lyophilized Test Vaccine. 

Use a sterile 1.0-mL syringe and an 18-gauge x 1 1/2-inch needle; mix by 

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

3.4.2 Dispense 1.8 ml MEM into each of 6, 12 x 75-mm polystyrene tubes 

labeled 10-1through 10-6.

3.4.3 With a micropipettor, transfer 200 μL from the rehydrated Test Vaccine

vial to the tube labeled 10-1; discard the pipette tip. Mix by vortexing.

3.4.4 Transfer 200 μL from the 10-1labeled tube (Section 3.4.3) to the 10-2tube; 

discard the pipette tip. Mix by vortexing.

3.4.5 Repeat Section 3.4.4 for each subsequent dilution, 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 Inoculate 5 wells/dilution of a CRFK Slide with 100 μL/well from dilutions 10-6

through 10-3of the Test Vaccine. 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., 

10-6through 10-3).

4.2 Inoculate 5 wells/dilution of a CRFK Slide with 100 μL/well, from dilutions 10-6

through 10-3of the CPV Working Positive Control. 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., 10-6through 10-3).

4.3 Five uninoculated wells serve as a Negative Cell Control.

4.4 Incubate CRFK Slides in a 36°± 2°C CO2 incubator for 5 days ± 1 day.

4.5 Following incubation, decant the media from the CRFK Slide and remove the 

plastic walls by twisting them away from the CRFK Slide, leaving the gasket attached to 

the slide.
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4.6 Place the CRFK Slides in a slide rack; place the rack in a glass staining dish filled 

with DPBS. Let stand for 15 ± 5 minutes at room temperature.

4.7 Discard the DPBS; fix the CRFK Slides in 100% acetone for 15 ± 5 minutes at 

room temperature. Remove and allow to air dry.

4.8 Pipette 75 ± 25 μL of the Working FPV Antiserum into each well of the CRFK 

Slides. Incubate for 30 ± 5 minutes in the aerobic incubator at 36°± 2°C.

4.9 Wash per Section 4.6.

4.10 Pipette 75 ± 25 μL of the Working Anti-species Conjugate into each well of the 

CRFK Slides. Incubate for 30 ± 5 minutes in the aerobic incubator at 36°± 2°C.

4.11 Wash per Section 4.6. Discard the DPBS.

4.12 Rinse the CRFK Slides with DI; allow to air dry.

4.13 Read at 100-200X magnification with a UV-light microscope; examine the cell 

monolayer for typical CPV apple-green nuclear fluorescence.

4.13.1 Wells containing 1 or more cells with specific CPV fluorescence are 

considered to be positive.

4.13.2 Results are recorded as number of IFA positive wells versus total number 

of wells examined for each dilution of a Test Vaccine and the CPV Working 

Positive Control.

4.14 Calculate the 50% fluorescent antibody infective dose (FAID50) of the Test 

Vaccine and the CPV Working Positive Control using the method of Spearman-Kärber as 

modified by Finney.

Example:

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

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

10-5 dilution of Test Vaccine = 2/5 wells IFA positive

10-6dilution of Test Vaccine = 0/5 wells IFA positive
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Spearman-Kärber formula:

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

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

d = log10 of tenfold dilution factor (1)

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

5 4 2 0 11

5 + 5 + 5 + 5 = 5 = 2.2

Test Vaccine titer = (3 – 1/2) + (1 • 2.2) = 4.7

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 CPV test 

vaccine, the recommended dose is 1 mL)

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

(for this CPV test 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 = 4.7 + 1.0 = 5.7

Therefore the titer of the CPV Test Vaccine is 105.7FAID50/mL.

5. Interpretation of the Test Results

5.1 Validity requirements:

5.1.1 The calculated FAID50 titer of the CPV Working 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.
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5.1.2 The lowest inoculated dilution of the CPV Working Positive Control must 

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

more wells are 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 IFA reaction (0/5).

5.1.3 The Uninoculated Cell Control must not exhibit any cytopathic effect, 

specific CPV fluorescence, 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 may be retested without prejudice.

5.3 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 

(APHIS) filed Outline of Production for the product under test, the Test Vaccine is 

considered SATISFACTORY.

5.4 If the validity requirements are met, but the titer of the Test Vaccine is less than 

the required minimum titer contained in the APHIS filed Outline of Production for the 

product under test, the Test Vaccine may be retested in accordance with the 9 CFR 113.8.

6. Report of Test Results

Report results as the FAID50 per dose of the Test Vaccine.

7. References

7.1 Title 9, Code of Federal Regulations, part 113.317, U.S. Government Printing 

Office, Washington, DC.

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

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

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

edition, pp. 394-401.
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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 in the Center for Veterinary 

Biologics and to provide additional detail. While no significant changes impacting the test were 

made from the previous protocol, the following changes have been made:

 2.1.2, 2.1.3 The temperature setting has been removed.

 2.1.6, 2.2.12, 2.2.13 Equipment has been added.

 2.2.3, 3.3.3 The reagent name has been corrected to “or CPV antiserum”.

 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.

 2.2.9, 3.3.4, 4.10 The conjugate name has been corrected to Anti-Species IgG.

 3.1 Personnel experience has been clarified.

 3.3.1 The cell count has been clarified.

 4.2 Inoculation of the Reference has been clarified.

 4.4 The incubation has been changed from hours to days.

 4.9, 4.11 The wash step has been corrected.
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 4.13.2 The recording step has been corrected.

 4.14 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.

 “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.

 The word “chamber” has been changed to “well” throughout the document.

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

individual items.

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