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SAM 117.05

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United States Department of Agriculture

Center for Veterinary Biologics

Testing Protocol

SAM 117

Supplemental Assay Method for Titration of Pseudorabies Virus Neutralizing 

Antibody (Constant Virus-Varying Serum Method)

Date: October 16, 2014

Number: SAM 117.05

Supersedes: SAM 117.04, September 9, 2011

Standard Requirement: 9 CFR 113.213

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

Peg A. Patterson

Approvals: /s/Geetha B. Srinivas Date: 26Nov14

Geetha B. Srinivas, Section Leader

Virology

/s/Byron E. Rippke Date: 09Dec14

Byron E. Rippke, Director

Policy, Evaluation, and Licensing

Center for Veterinary Biologics

/s/Rebecca L.W. Hyde Date: 10Dec14

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 Pseudorabies Virus Neutralizing Antibody

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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

Appendices
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Supplemental Assay Method for the Titration of Pseudorabies Virus Neutralizing Antibody

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1. Introduction

This Supplemental Assay Method (SAM) describes an in vitro serum neutralization (SN) test 

which utilizes cytopathic effects (CPE) or fluorescent antibody (FA) technique in a cell culture 

system to determine the SN antibody titer against pseudorabies virus (PRV). The SN assay uses a 

constant amount of virus to test varying dilutions of serum. The assay meets the requirements in 

title 9, Code of Federal Regulations (9 CFR), to test serum samples collected from vaccinated 

and control swine for potency testing of inactivated PRV vaccines.

2. Materials

2.1 Equipment/instrumentation

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

below.

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

Scientific Inc.)

2.1.2 2 water baths

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

2.1.4 Fluorescent microscope (Model BH2, Olympus America Inc.)

2.1.5 96-well cell culture plates

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

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

2.1.8 12 x 75-mm polystyrene tubes

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 PRV reference virus, Shope strain
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2.2.2 Madin-Darby bovine kidney (MDBK) or other permissive cells free of 

extraneous agents as tested by 9 CFR

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)

3. Q.S. to 1000 mL with deionized water (DI), adjust pH to 6.8-6.9 with 

2N hydrochloric acid (HCl)

4. Aseptically add 5 mL lactalbumin hydrolysate

5. Sterilize through 0.22-μm filter.

6. Aseptically add:

a. 10 mL L-glutamine (200 mM)

b. 50 μg/mL gentamicin sulfate

7. Store at 2°- 7°C.

2.2.4 Growth Medium

1. 900 mL of MEM

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

2.2.5 Maintenance Medium

1. 98 mL of MEM

2. 2 mL of FBS

2.2.6 Swine Anti-Pseudorabies Fluorescein Isothiocyanate Labeled Conjugate

2.2.7 0.01 M Phosphate buffered saline (PBS) (NCAH Media #30054)

1. 1.19 g sodium phosphate, dibasic, anhydrous (Na2HPO4)

0

2. 0.22 g sodium phosphate, monobasic, monohydrate (NaH2PO4•H2O)
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3. 8.5 g sodium chloride (NaCl)

4. Q.S. to 1000 mL with distilled water 

5. Adjust pH to 7.2-7.6 with 0.1 N sodium hydroxide (NaOH) or 1.0 N 

hydrochloric acid (HCl).

6. Sterilize by autoclaving at 15 psi, 121°± 2°C for 35 ± 5 minutes.

7. Store at 2°- 7°C.

2.2.8 80% Acetone

1. 80 mL acetone

2. 20 mL distilled water

3. Store at room temperature.

2.2.9 A PRV antibody negative control serum

2.2.10 A PRV antibody positive control serum

3. Preparation for the Test

3.1 Personnel qualifications/training

Personnel must have training in the immunologic basis of SN assays, cell culture 

techniques, FA test techniques, and in the principles of aseptic techniques.

3.2 Preparation of equipment/instrumentation

3.2.1 Set a water bath at 56°± 2°C.

3.2.2 Set a 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 cell culture plates with 

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

after 2 days of incubation at 36°± 2°C. This becomes the MDBK Plate. Growth 

Medium is changed if excess acidity of the medium is observed as indicated by a 
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change from red to yellow of Growth Medium or cells are not confluent 2 days 

after incubation.

3.3.2 On day of test performance

1. Working Virus Preparation. Rapidly thaw a vial of PRV Reference 

Virus in a 36°± 2°C water bath. Dilute the virus in MEM to contain 50-

300 50% tissue culture infective dose (TCID50)/25 μL.

See Appendix II for titration of PRV and determination of the dilution 

containing the optimal virus dose for the test.

2. Virus Back Titration. Make 3 serial tenfold dilutions of Working 

Virus.

a. Place 0.9 mL of MEM into 3, 12 x 75-mm polystyrene tubes 

labeled 10-1to 10-3respectively.

b. Transfer 0.1 mL of Working Virus to the 10-1tube; mix by 

vortexing. Discard pipette tip.

c. Transfer 0.1 mL from the 10-1tube to the 10-2tube; mix by 

vortexing. Discard pipette tip.

d. Repeat Step 2.c to the remaining tube, transferring from the 10-2

to the 10-3tube.

3. On the day of MDBK Plate examination, dilute Swine Antipseudorabies Fluorescein Isothiocyanate Labeled Conjugate according to 

manufacturer’s instructions.

3.4 Preparation of the sample (on day of test performance)

3.4.1 Heat inactivate all Test Serum Samples, Negative Control serum and the 

Positive Control serum in a 56°± 2°C water bath for 30 ± 5 minutes.

3.4.2 Prepare serial twofold dilutions of Test Serum Samples, Negative Control 

serum, and Positive Control serum in a 96-well cell culture plate, which becomes 

the Transfer Plate (see Appendix I). Twofold dilutions are made as follows:

1. Add 150 μL MEM to all wells in Rows B-H.
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2. Add 150 μL Test Serum Samples, Positive Control serum, and 

Negative Control serum to Rows A and B. Mix Row B with the multichannel micropipettor (4-5 fills). The same tips may be used throughout.

3. Transfer 150 μL from Row B to Row C. Mix Row C with the multichannel micropipettor (4-5 fills).

4. Continue as in Step 3 for the remaining rows. Discard 150 μL from all 

wells in Row H.

5. Add 150 μL of Working Stock Virus to all wells of the Dilution Plate. 

Tap plates gently to mix. Incubate for 60 ± 10 minutes at 36°± 2°C to 

allow for neutralization of virus. The addition of virus is considered to be 

an additional twofold dilution of Test Serum Samples.

4. Performance of the Test

4.1 Decant Growth Medium from a MDBK Plate.

4.2 Inoculate 50 μL/well of each Virus-Test Serum or Virus-Control Serum mixture 

into 5 wells/dilution of a MDBK Plate.

4.3 Inoculate 25 μL of each dilution of Virus Back Titration into 5 wells of a MDBK 

Plate. Add an additional 25 μL of MEM to all wells of the Back Titration.

4.4 Maintain 2 or more wells on a MDBK Plate as uninoculated cell controls.

4.5 Incubate MDBK Plates for 60 ± 10 minutes at 36°± 2°C.

4.6 Add 200 μL/well of Maintenance Medium to all wells (do not remove virus serum 

mix). Incubate MDBK Plate for 4 days ± 1 day postinoculation at 36°± 2°C.

4.7 CPE counting is the primary method of determining the TCID50.

4.7.1 At four days postinoculation, examine the wells with an inverted 

microscope. The CPE of PRV is visible as grape-like clusters of rounded cells in 

the cell monolayer where the cells have been destroyed by the virus.

4.7.2 Record the number of wells/dilution showing any characteristic CPE of 

PRV for each Test Serum Sample and Virus Back Titration.

4.7.3 Calculate the TCID50 of the Working Dilution of virus through the Virus 

Back Titration using the Spearman-Kärber method as modified by Finney.
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Example: 

10-0dilution of Working Virus = 5 of 5 wells CPE Positive

10-1dilution of Working Virus = 5 of 5 wells CPE Positive

10-2dilution of Working Virus = 3 of 5 wells CPE Positive

10-3dilution of Working Virus = 0 of 5 wells CPE Positive

Titer = (X - d/2 + [d • S]) where:

X = Log10 of lowest dilution (=0)

d = Log10 of dilution factor (=1)

S = Sum of proportion of CPE Positive

5 5 3 0 13

5 + 5 + 5 + 5 = 5 = 2.6

Titer = (0 – 1/2 + [1 • 2.6] = 2.1

Antilog of 2.1 = 125.9

Titer of the Working PRV is 125 TCID50/25 μL dose in the test.

4.7.4 Calculate the endpoint for each Test Serum, Positive Control serum and 

Negative Control serum using the Spearman-Kärber method. The endpoints of 

these sera are reported as SN titer which corresponds to the reciprocal of the 

highest serum dilution that neutralizes the reference PRV.

Example:

1:2 dilution of Test Serum = 5 of 5 wells CPE Negative

1:4 dilution of Test Serum = 5 of 5 wells CPE Negative

1:8 dilution of Test Serum = 3 of 5 wells CPE Negative

1:16 dilution of Test Serum = 0 of 5 wells CPE Negative

Titer = (X - d/2 + [d • S]) where:

X = Log10 of lowest dilution (=0.3)

d = Log10 of dilution factor (=0.3)

S = Sum of proportion of CPE negative 

5 5 3 0 13

5 + 5 + 5 + 5 = 5 = 2.6
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Supplemental Assay Method for the Titration of Pseudorabies Virus Neutralizing Antibody

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Titer = (0.3 – 0.3/2 + [0.3 • 2.6] = 0.93

Antilog of 0.93 = 8.5

Titer of the Test Serum = 9 

4.8 If CPE is difficult to interpret, an FA Test may be conducted as follows:

4.8.1 Decant media from the MDBK Plate.

4.8.2 Fill wells with 80% acetone.

4.8.3 Incubate at room temperature for 15 ± 5 minutes.

4.8.4 Decant the 80% acetone from the MDBK Plate and air dry at room 

temperature.

4.8.5 Pipette 60 ± 10 μL of Swine Anti-Pseudorabies Fluorescein Isothiocyanate 

Labeled Conjugate into all wells. Incubate for 45 ± 15 minutes at room 

temperature.

4.8.6 Rinse by filling the wells completely in PBS allow to stand for 5 ± 2 

minutes, and decant.

4.8.7 Repeat for a total of 2 washes, decant, and allow to air dry or dry at 

36°± 2°C.

4.8.8 Examine wells with a fluorescent microscope.

4.8.9 A well is considered positive if typical nuclear, apple-green fluorescence 

is observed.

4.8.10 Record and calculate as in Section 4.7.4.

5. Interpretation of the Test Results

5.1 The test is invalid if visible contamination or serum toxicity is observed in wells 

of any dilution of a Test Serum Sample.

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

5.3 For a valid assay, the Virus Back Titration must have between 50-300 TCID50/

25 μL.
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5.4 The Positive Control serum titer must be within the value of the mean titer ± one 

dilution.

5.5 The Negative Control serum must be negative at the 1:2 dilution

6. Report of Test Results

Record all test results on the test record.

7. References

7.1 Title 9, Code of Federal Regulations, part 113.213, U.S. Government Printing 

Office, Washington, DC.

7.2 Banks M, Cartwright S. Comparison and evaluation of four serological tests for 

detection of antibodies to Aujeszky's disease virus. Vet Rec (1983) 113:38-41.

7.3 Conrath TB.: Handbook of Microtiter Procedures. 1972. Clinical and Research 

Applications Laboratory, Cooke Engineering Company, Alexandria, Virginia.

7.4 Finney DJ. Statistical Method in Biological Assay. 3rd ed. 1978. Charles Griffin 

and Company, London, pp. 394-401.

7.5 Geval SH, Joo HS, Mourning JR, McPherson SW, Goval K. Comparison of three 

serotests for the detection of pseudorabies antibodies in pigs. Comp Immunol Microbiol 

Infect Dis (1987) 10(3-4):67-71.

7.6 Oren SL, Swenson SL, Kinker DR, Hill HT, et al. Evaluation of serological 

pseudorabies tests for the detection of antibodies during early infections. J Vet Diag 

Invest (1993) 5(4):529-33.

7.7 Parker RA, MA Pallansch. Using the virus challenge dose in the analysis of virus 

neutralization assays. Statistics in Medicine, (1992) 11:1253-1262.

7.8 Rose NR, Friedman H, Fahey JL, eds. Manual of Clinical Laboratory 

Immunology. Chapter 11: Neutralization Assays. 1986. ASM, Washington, D.C.

7.9 Weir DM, Ed. Handbook of Experimental Immunology. Volume 3. Application of 

Immunological Methods. Blackwell Scientific Publications, Edinburgh, England. Chapter 

40: Immunological Methods in Virology.
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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 Contact information has been updated.

 The document number has been changed from VIRSAM0117 to SAM 117.

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:

 2.2.3.2 The formulation of MEM was altered to adjust the concentration of sodium 

bicarbonate to 1.1 grams/liter from 2.2 grams. Penicillin, streptomycin, and 

amphotericin B were removed from the formulation.

 2.2.7 Phosphate buffered saline (PBS) has been added.

 2.2.9 PRV antibody negative control serum has been added.

 2.2.10 PRV antibody positive control serum has been added.

 3.3.2.1 Appendix II has been added.

 3.4.1/3.4.2 Positive and Negative Control serums have been added.

 4.3 50 μl dilution of Virus Back Titration has been changed to 25 μl. The addition of 

25 μl of MEM has also been added. 

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

Spearman-Kärber formula.
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 5.4/5.5 Added Positive and Negative Control titer interpretation guidelines.

 7.1 Added reference to the Code of Federal Regulations (9CFR).

 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.
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Appendix I

Transfer Plate

1 2 3 4 5 6 7 8 9 10 11 12

A 

1:2

TS1 TS1 TS1 TS1 TS1 CC CC TS2 TS2 TS2 TS2 TS2

B 

1:4

C 

1:8

D 16

E 32

F 64

G 

128

H 

256

TS= Test Serum

CC= Cell Control
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Appendix II

Titration of PRV and determination of the dilution containing the optimal virus dose for the Beta 

SN test

1. Label 8, 17 x 100-mm plastic tubes by identifying 1 tube as 10-1, 1 tube for 10-2, etc.

through 10-8. The number of tubes required will depend on the endpoint of the reference 

virus.

2. Dispense 4.5 mL of cell culture medium in each of the tubes.

3. Rapidly thaw, using a 36°C water bath, one vial of Reference cytopathic BVD 

virus, and using a pipette or pipettor and tip, transfer 0.5 mL of virus to the first tube 

(10-1).

4. Discard pipette or pipettor tip, and vortex the 10-1virus dilution tube.

5. Using a new pipette or pipettor tip, transfer 0.5 mL of the 10-1dilution of virus to 

the 10-2tube, discard pipette or pipettor tip, and vortex the 10-2dilution. Continue 

diluting in a similar fashion through the last tube, using a new pipette or pipettor tip for 

each transfer.

6. Transfer 0.025 mL of each virus dilution to each of 5 wells of a MDBK cell plate as 

prepared in Section 3.3.1. The plate is then incubated in a 36o± 2C, 5% CO2 incubator 

for 4 to 5 days.

7. The titration plate is read for CPE, and the results are recorded as number of wells 

exhibiting CPE per number of wells inoculated with each virus dilution.

8. Using the Spearman-Kärber method, calculate the endpoint of the PRV virus 

titration. The titer is expressed as log10 tissue culture infective doses fifty (TCID50) per 

25 μL dose.

Example:

10-1dilution PRV = 5 of 5 wells CPE Positive

10-2dilution PRV = 5 of 5 wells CPE Positive

10-3dilution PRV = 5 of 5 wells CPE Positive

10-4dilution PRV = 5 of 5 wells CPE Positive

10-5dilution PRV = 3 of 5 wells CPE Positive

10-6dilution PRV = 0 of 5 wells CPE Positive
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Titer = (X - d/2 + [d • S]) where:

Titer = (1 - 1/2 + [1 • 4.6]) = 5.1

The antilog of 5.1 = 125893

Therefore, a 1:125,893 dilution of the virus should yield a virus concentration of 1 

TCID50 per 25 μL. Assuming a 200 TCID50 per 25 μL is the optimal dosage of 

virus to use in the SN test, divide the 125,893 by 200 to obtain 629.46. The 

dilution factor to obtain 200 TCID50 per 25 μL would then be 1:629. This 

Working Reference PRV dilution must be confirmed by performing a back 

titration. A series of 3 to 5 dilutions of virus bracketing the expected dilution may 

be tested with back titrations of each. Adjustments from the original calculated 

dilution may need to be made to obtain the correct working virus dose.