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SAM 114.05

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United States Department of Agriculture

Center for Veterinary Biologics

Testing Protocol

SAM 114

Supplemental Assay Method for Titration of Porcine Transmissible 

Gastroenteritis Virus

Date: October 16, 2014 Approved, Pending Standard Requirement

Number: SAM 114.05

Supersedes: SAM 114.04, September 9, 2011

Standard Requirement:

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

Peg A. Patterson,

Approvals:

/s/Geetha B. Srinivas _ Date: 10Dec14

Geetha B. Srinivas, Section Leader

Virology

/s/Byron E. Rippke Date: 16Dec14

Byron E. Rippke, Director

Policy, Evaluation, and Licensing

Center for Veterinary Biologics

/s/Rebecca L.W. Hyde Date: 16Dec14

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 Porcine Transmissible Gastroenteritis 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 (on day of test performance)

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 assay method which utilizes 

cytopathic effect (CPE) in a cell culture system to determine viral titers of modified live porcine 

transmissible gastroenteritis (TGE) virus vaccines.

2. Materials

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

below.

2.1 Equipment/instrumentation

2.1.1 36°± 2°C, 5% ± 1% CO2, high-humidity incubator (Model 3158, Forma 

Scientific Inc.)

2.1.2 Water bath

2.1.3 Inverted microscope (Model CK, Olympus America Inc.)

2.1.4 96-well microtiter plates

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

2.1.6 Micropipettors: 200-μL and 1000-μL single channel; 300-μL x 12-channel

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 TGE Reference Virus, Purdue Strain

2.2.2 Swine testicular (ST) cells free of extraneous agents as tested by title 9,

Code of Federal Regulations (9 CFR), part 113.52 (available from Center for 

Veterinary Biologics)

2.2.3 Minimum Essential Medium (MEM) (National Centers for Animal 

Health (NCAH) Media #20030)

1. 9.61 g MEM with Earles salts without bicarbonate

2. 1.1 g sodium bicarbonate (NaHCO3)
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3. Q.S. to 1000 mL with deionized water (DW), adjust pH to 6.8-6.9 with 

2N hydrochloric acid (HCl).

4. Sterilize through 0.22-μm filter.

5. Aseptically add:

a. 10 mL L-glutamine (200 mM)

b. 5 mL lactalbumin hydrolysate or edamine

c. 50 μg/mL gentamicin sulfate

6. Store at 2°- 7°C.

2.2.4 Growth Medium

1. 900 mL MEM

2. Aseptically add 100 mL gamma-irradiated fetal bovine serum (FBS).

3. Store at 2°- 7°C.

2.2.5 Dilution Medium

1. 98 mL of MEM

2. 2 mL of FBS

3. 2% sodium pyruvate

4. Store at 2°- 7°C

2.2.6 12 x 75-mm polystyrene tubes

2.2.7 Self-refilling, 2-mL repetitive syringe

2.2.8 Graduated cylinders 25-, 50-, 100-, and 250-mL
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3. Preparation for the Test

3.1 Personnel qualifications/training

Personnel must have experience in the basis of cell-culture techniques, virus titration assays, 

and in the principles of aseptic techniques.

3.2 Preparation of equipment/instrumentation

Set the water bath at 36°± 2°C

3.3 Preparation of reagents/control procedures

3.3.1 Two days prior to test performance: Seed 96-well microtiter plates with 

ST cells, in Growth Medium, at a cell count that will produce a monolayer 

after 2 days of incubation at 36°± 2°C. These become the ST plates. Growth 

Medium is changed if excess acidity of the medium is observed or cells are 

not confluent 2 days after incubation.

3.3.2 On day of test performance

1. Rapidly thaw a vial of TGE Reference Virus in 36°± 2°C water bath.

2. Reference Virus titration

Make 5 serial tenfold dilutions of Reference Virus as follows:

a. Dispense 1.8 mL of Dilution Medium into 5, 12 x 75-mm polystyrene 

tubes labeled 10-1to 10-5respectively using a repetitive syringe.

b. Transfer 200 μL of Reference Virus to the 10-1tube; mix by 

vortexing. Discard pipette tip.

c. Transfer 200 μL from the 10-1tube to the 10-2tube; mix by vortexing. 

Discard pipette tip.

d. Repeat Step 2.c to the remaining tubes, transferring 200 μL from 

previous dilution to the next dilution.
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3.4 Preparation of the Test Vaccine (on day of test performance)

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 test initiation, remove the seal and stopper from both the 

Test Vaccine and the bottle containing the accompanying diluent. Measure the 

diluent into a sterile graduated cylinder according to the Test Vaccine’s total volume 

indicated on the manufacturer’s label (e.g., for a 100-dose container of 2-mL-perdose, reconstitute with 200 mL of diluent). Aseptically pour the diluent into the 

lyophilized bottle of vaccine. Mix by vortexing. Porcine rotavirus, if present in the 

test vaccine, is not usually blocked with specific antisera due to its poor growth on ST 

cells in the absence of proteolytic enzymes.

Note: If the vaccine being evaluated is a bivalent TGE/Porcine rotavirus 

product, it is not necessary to neutralize the Porcine rotavirus fraction due to its 

poor growth on ST cells in the absence of proteolytic enzymes.

3.4.2 Prepare serial tenfold dilutions of Test Vaccine. Serial tenfold dilutions 

may be made as follows:

1. Dispense 1.8 mL of Diluent Medium into labeled tubes using 

repetitive syringe.

2. Pipette 200 μL of Test Vaccine to the 10-1tube, mix by vortexing. 

Discard pipette tip.

3. Repeat Step 2 to the remaining tubes transferring 200 μL from previous 

dilution to the next dilution. Continue as needed (10-2to 10-5).

4. Performance of the Test

4.1 Decant the Growth Media from ST plates.

4.2 Pipette 200 μL/well from each Test Vaccine tube to 5 wells on an ST plate.

4.3 Pipette 200 μL/well of each Reference Virus titration tube to 5 wells on an ST plate.

4.4 Maintain 5 or more wells as uninoculated cell-culture controls.

4.5 Incubate the ST plates undisturbed at 36°± 2°C in the CO2 incubator for 4 days ± 12 

hours.

4.6 After 4 days ± 12 hours inoculation, examine the wells with an inverted microscope. 

The CPE of TGE is visible as cellular death in the cell monolayer where the cells have been 

destroyed by the virus.
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4.6.1 Record the number of wells/dilution, showing any characteristic CPE of TGE 

for each Test Vaccine and Reference Virus Titration.

4.6.2 The 50% tissue culture infective dose (TCID50) of the Test Vaccine and 

Reference Virus Titration are calculated using the Spearman-Kärber method as 

modified by Finney. The titers are expressed as log10 TCID50 per dose.

Example:

10-3dilution of vaccine = 5/5 wells CPE positive

10-4dilution of vaccine = 4/5 wells CPE positive

10-5dilution of vaccine = 1/5 wells CPE positive

10-6dilution of vaccine = 0/5 wells CPE positive

Spearman-Kärber formula:

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

X = log10 of dilution with all wells infected (3)

d = log10 of dilution factor (1)

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

5 4 1 0

5 = 1.0 + 5 = 0.8 + 5 = 0.2 + 5 = 0 = 2.0

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

Adjust the titer to the recommended Test Vaccine dose as follows:

1. Divide the Test Vaccine Dose by the Inoculation Dose

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

this test vaccine, the recommended dose is 2 mL)

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

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

 2 mL dose = 10

 0.2 mL inoculum

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

below:

Log of 10 = 1.0
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Test Vaccine titer = 4.5 + 1.0 = 5.5

Therefore the titer of the Test Vaccine is 105.5TCID50/2 mL

5. Interpretation of the Test Results

5.1 The test is invalid if visible contamination is observed.

5.2 The test is invalid if CPE is observed in any of the control wells.

5.3 For a valid assay, the Reference Virus Titration 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.

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

and can be retested without prejudice.

5.5 If the validity requirements are met and the titer of the vaccine is greater than or equal 

to the titer contained in the approved Outline of Production for the product under test, the 

product is considered SATISFACTORY.

5.6 If the validity requirements are met but the titer of the Test Vaccine is lower than the 

required minimum, it must be retested according to 9 CFR 113.8.

6. Report of Test Results

Record all test results on the test record.

7. References

7.1 Title 9, Code of Federal Regulations, U.S. Government Printing Office, Washington, 

DC.

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

microbiology. Comstock Publishing Associates, a division of Cornell University 

Press/Ithaca and London, pg 731.

7.3 Finney, D.J. 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 phrase "available from the Center for Veterinary Biologics/CVB" has been 

removed from the document as these reagents are no longer supplied by the CVB.

Version .03

 The document number has been changed from VIRSAM0114 to SAM 114.

 The Contact information has been updated.

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

1.1 g. Penicillin, streptomycin and amphotericin B have been removed from the 

media formulation.

 3.4.1 This has been rewritten from “reconstituting with syringe and needle” to 

“graduated cylinder”. Also that a test is from a single vial.

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

Spearman-Kärber formula.

 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.