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SAM 305.05

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United States Department of Agriculture

Center for Veterinary Biologics

Testing Protocol

SAM 305

Supplemental Assay Method for the Titration of Feline Panleukopenia Virus 

in Cell Culture

Date: October 30, 2014

Number: SAM 305.05

Supersedes: SAM 305.04, June 30, 2011

Standard Requirement: 9 CFR 113.304

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 the Titration of Feline Panleukopenia Virus in 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 the Titration of Feline Panleukopenia Virus in Cell Culture

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1. Introduction

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

modified-live feline panleukopenia virus (FPV) vaccines for viral content. The method uses the 

Crandall feline kidney (CRFK) cell line as the test system. Presence or absence of FPV is 

determined by staining inoculated cell cultures by an indirect fluorescent antibody (IFA) 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 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 Micropipettor and/or motorized microliter pipette and tips

2.1.7 Glass slides, 8-chamber (Lab-Tek®slides)

2.1.8 Glass staining dish with rack for Lab-Tek slides (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 FPV Positive Control, ICK 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 FPV 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. Q.S. to 1000 mL with deionized water (DI); adjust pH to 6.8-6.9 with 

2N hydrochloric acid (HCl).

4. Sterilize through a 0.22-μm filter.

5. Aseptically add 50 μg/mL gentamicin sulfate

6. Store at 2°- 7°C.

2.2.5 Growth Medium

1. 920 mL 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 in 900 mL DI.

6. Dissolve 1.03 g sodium phosphate, dibasic, anhydrous (Na2HPO4) 

with 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.
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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 Polystyrene tubes, 12 x 75-mm

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

conjugate (Anti-species Conjugate)

2.2.9 100% acetone

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

2.2.11 Pipette-aid

2.2.12 Disposable transfer pipette, 3.5-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 IFA.

3.2 Preparation of equipment/instrumentation

3.2.1 On the day of initial titration, set a water bath at 56°± 2°C.

3.2.2 On the day of initial titration, set a water bath at 36°± 2°C.

3.2.3 On the day of the IFA test, 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 Preparation of CRFK slides

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

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

0.4 mL/chamber of approximately 105.2 ± 104.9 cells/mL diluted in Growth 

Medium into all chambers of the Lab-Tek®Slides. Prepare sufficient LabTek®

Slides to allow 25 chambers for controls and 20 chambers for each 

Test Vaccine. Incubate at 36°± 2°C in a CO2 incubator and use within 4 

hours. These become the CRFK Slides.
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2. Use seeded CRFK Slides within 4 hours.

3.3.2 Preparation of Working FPV Positive Control

1. On the day of inoculation, rapidly thaw a vial of FPV 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 FPV Positive Control to the tube labeled 10-1; 

discard pipette tip. Mix by vortexing.

4. Transfer 200 μL from the 10-1labeled tube to the 10-2tube; discard 

pipette tip. 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 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 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.

3.4 Preparation of the Test Vaccine

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 1-mL-dose vaccine, 0.5 mL for 1/2-mL-dose vaccine, 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 For a monovalent FPV Test Vaccine, mix 200 μL of Test Vaccine with 

1.8 mL of MEM in a 12 x 75-mm polystyrene tube for a 10-1dilution. Mix by 

vortexing.
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3.4.3 For a multifraction Test Vaccine containing FPV, heat inactivate the nonFPV fraction(s) in the Test Vaccine in a 56°± 2°C water bath for 60 ± 5 minutes. 

Dilute according to Section 3.4.2.

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

labeled 10-1through 10-6.

3.4.5 Transfer 200 μL from the Rehydrated Test Vaccine to the tube labeled

10-1; discard pipette tip. Mix by vortexing.

3.4.6 Transfer 200 μL from the tube labeled 10-1to the tube labeled 10-2; discard 

pipette tip. Mix by vortexing.

3.4.7 Repeat Section 3.4.6 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 chambers/dilution of a CRFK Slide with 100 μL/chamber from 

dilutions 10-6through 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 chambers/dilution of a CRFK Slide with 100 μL/chamber, from 

dilutions 10-6through 10-3of the Working FPV 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 chambers serve as negative cell controls.

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 wall by twisting them away from the CFRK Slide, leaving the gasket attached to 

the CRFK Slide.

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

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

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

room temperature. Remove and allow to air dry.
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4.8 Pipette 75 ± 25 μL of the Working FPV Antiserum into each chamber of the 

CRFK Slides with a transfer pipette. Incubate in the aerobic incubator at 36°± 2°C for 

30 ± 5 minutes.

4.9 Wash per Section 4.6. Discard the DPBS.

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

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

4.11 Wash per Section 4.6. Discard the DBPS.

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

4.13 Read at 100-200X with a UV-light microscope. Examine the cell monolayer for 

typical apple-green nuclear fluorescence.

4.13.1 Chambers containing one or more cells displaying specific fluorescence 

for FPV are positive.

4.13.2 Results are recorded as the number of IFA positive chambers versus total 

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

Working FPV Positive Control.

4.14 Calculate the FPV endpoints of the Test Vaccine and the Working FPV Positive

Control using the Spearman-Kärber method as commonly modified by Finney. The titer 

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

Example:

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

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

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

10-6dilution 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 IFA positive (3) 

d = log10 of tenfold dilution factor (1)

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

5 4 2 0 11

5 + 5 + 5 + 5 = 5 = 2.2
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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 

FPV test vaccine, the recommended dose is 1 mL) 

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

Test Slide (for this FPV 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 FPV Test Vaccine is 105.7 FAID50/mL.

5. Interpretation of the Test Results

Validity requirements:

5.1 The calculated titer of the Working FPV 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 uninoculated cell controls must not exhibit any cytopathic effect, specific 

FPV fluorescence, or cloudy media that would indicate contamination.

5.3 The lowest inoculated dilution of the FPV Positive Control must produce a FA 

positive reaction in 100% of the chambers (5/5). If an endpoint is not reached, (1 or more 

chambers are FA 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 reaction (0/5).

5.4 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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5.5 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 

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

considered SATISFACTORY.

5.6 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 is retested in accordance with 9 CFR 113.8.

6. Report of Test Results

Results are reported as the FAID50 per dose of Test Vaccine.

7. References

7.1 Title 9, Code of Federal Regulations, part 113.304, U.S. Government Printing 

Office, Washington, DC.

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

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

7.3 Finney, D.J. 1978. Statistical methods 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.
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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 Removed self-refilling syringe from the list of Reagents/supplies needed.

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

to 1.1 g.

 2.2.4.5 Penicillin and streptomycin have been deleted.

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

Spearman-Kärber formula. 

 5.1.3 Clarification of the endpoints for a valid assay has been added.

 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.

 “Tissue culture infective dose (TCID50)” has been changed to “fluorescent antibody 

infective dose (FAID50)” throughout the document.

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

individual items.