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SAM 115.03

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United States Department of Agriculture

Center for Veterinary Biologics

Testing Protocol

SAM 115

Supplemental Assay Method for Titration of Bovine Virus Diarrhea 

Neutralizing Antibody (Constant Serum-Varying Virus Method)

Date: October 16, 2014

Number: SAM 115.03

Supersedes: SAM 115.02, February 9, 2011

Standard Requirement: 9 CFR 113.146

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 Titration of Bovine Virus Diarrhea 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 Titration of Bovine Virus Diarrhea Neutralizing Antibody 

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1. Introduction

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

to determine the neutralizing activity against Bovine Virus Diarrhea (BVD) type 1 and 2 virus.

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, 5% ± 1% CO2, high humidity 

2.1.2 Vortex mixer

2.1.3 Microscope, inverted light

2.1.4 Microscope, fluorescent

2.1.5 Micropipettor, 200-μL, 1000-μL single channel, 5- to 50-μL x 12-channel,

and tips

2.1.6 Water bath

2.1.7 Centrifuge and rotor

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 Indictor Virus

1. BVD type 1

2. BVD type 2

2.2.2 Bovine Turbinate (BT) cell cultures free of extraneous agents as tested by 

title 9, Code of Federal Regulations (9 CFR).
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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. 2.2 g sodium bicarbonate (NaHCO3) 

3. Dissolve Steps 1 and 2 with 900 mL deionized water (DI).

4. Add 5 g lactalbumin hydrolysate or edamine to 10 mL DI. Heat to 

60°± 2°C until dissolved. Add to Step 3 with constant stirring.

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:

a. 10 mL L-glutamine

b. 50 μg/mL gentamicin sulfate

8. Store at 4°± 2°C.

2.2.4 Growth Medium

1. 900 mL of MEM

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

3. Store at 4°± 2°C.

2.2.5 Monoclonal antibodies (MAb)

1. Anti-BVD type 1 MAb

2. Anti-BVD type 2 MAb

2.2.6 Anti-mouse fluorescein isothiocyanate labeled conjugate (Anti-Mouse

Conjugate)
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2.2.7 80% Acetone

1. 80 mL acetone

2. 20 mL DI

3. Store at room temperature (23°± 2°C).

2.2.8 0.01 M Phosphate buffered saline (PBS) (NCAH Media #30054)

1. 1.33 g sodium phosphate, dibasic, anhydrous (Na2HPO4)

2. 0.22 g sodium phosphate, monobasic, monohydrate (NaH2PO4•H2O)

3. 8.5 g sodium chloride (NaCl)

4. Q.S. to 1000 mL with DI.

5. Adjust pH to 7.2-7.6 with 0.1 N sodium hydroxide (NaOH) or 2N HCl.

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

7. Store at 4°± 2°C.

2.2.9 FBS negative for BVD antibodies

2.2.10 Cell culture plate, 96-well

2.2.11 Polystyrene tube, 17 x 100-mm

2.2.12 Serological pipette, 10-mL

2.2.13 Plastic wash bottle, 500-mL

3. Preparation for the Test

3.1 Personnel qualifications/training

Personnel must have training in antibody titration assays, cell culture maintenance, and in 

the principles of aseptic techniques.
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3.2 Preparation of equipment/instrumentation

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

3.2.2 On day of test initiation, set a water bath at 56°± 2°C.

3.3 Preparation of reagents/control procedures

3.3.1 One day prior to test initiation, seed 96-well cell culture plates with BT 

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

after 1 day of incubation at 36°± 2°C in a CO2 incubator. These become the BT 

Test Plates.

3.3.2 On day of test initiation, rapidly thaw a vial of an appropriate Indicator 

Virus in a 36°± 2°C water bath. The number of dilutions depends upon the 

predetermined titer of the Indicator Virus. Prepare serial tenfold dilutions as 

follows:

1. Pipette 9.0 mL of MEM with a serological pipette into 8, 17 x 100-mm 

polystyrene tubes labeled 10-1to 10-6.

2. Transfer 1.0 mL of an Indicator Virus to the 10-1tube; mix by 

vortexing. Discard the pipette.

3. Transfer 1.0 mL from the 10-1tube to the 10-2tube; mix by vortexing. 

Discard the pipette.

4. Repeat Step 3 for each of the subsequent dilutions, transferring 1.0 mL

from the previous dilution to the next dilution.

3.3.3 On the day of BT Test Plate examination, dilute Anti-BVD MAb and 

Anti-Mouse Conjugate in PBS according to the supplied Center for Veterinary 

Biologics (CVB) Reference and Reagent Data Sheet.

3.4 Preparation of the sample

On day of, or prior to test initiation, heat inactivate all Test Sera in a 56°± 2°C water bath 

for 30 ± 5 minutes.
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4. Performance of the Test

4.1 Add 150 μL/well of an undiluted Test Serum into a column of a 96-well cell 

culture plate, which becomes the Dilution Plate (Appendix I).

4.2. Add 150 μL/well of Reference Serum (if used) into a column of a Dilution Plate.

4.3 Add 150 μL/well of the last 4 dilutions of the Indicator Virus to a row of the 

Dilution Plate.

4.4 Mix by tapping the edge of the Dilution Plate with fingers. Incubate for 60 ± 10 

minutes at 36°± 2°C to allow for neutralization of the Indicator Virus.

4.5 Inoculate 50 μL/well of each Virus-serum mixture into 5 wells/dilution of the 

appropriate Test Plate (Appendix II).

Note: The Growth Media is not removed from the BT Test Plates prior to 

inoculation.

4.6 Maintain 5 or more wells on each Test Plate as uninoculated cell controls.

4.7 Inoculate 25 μL/well of the last 4 dilutions of the Indicator Virus to 5 

wells/dilution as an endpoint titration.

4.8 Incubate the BT Test Plates for 4 days ± 12 hours postinoculation at 36°± 2°C in a 

CO2 incubator.

4.9 At the end of the incubation period, examine the wells with an inverted light 

microscope. Record the number of wells/dilution showing any characteristic CPE for 

each Test Sera and for the Reference Serum.

4.10 Alternatively, an indirect fluorescent antibody technique (IFA) may be conducted 

to determine the SN titer against BVD as follows:

4.10.1 Decant media from the BT Test Plates.

4.10.2 Fill wells with 80% acetone.

4.10.3 Incubate at room temperature for 15 ± 5 minutes.

4.10.4 Decant the 80% acetone from the BT Test Plate; air dry at room 

temperature.
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4.10.5 Pipette 50 μL/well of a diluted Anti-BVD MAb into a BT Test Plate; 

incubate for 45 ± 15 minutes at 36°± 2°C.

4.10.6 Rinse by filling the wells completely with PBS; decant the liquid.

4.10.7 Repeat Step 6 for a total of 2 washes.

4.10.8 Pipette 50 μL/well of the diluted Anti-mouse Conjugate into the BT Test 

Plates; incubate for 45 ± 15 minutes at 36°± 2°C.

4.10.9 Repeat Step 6 for a total of 2 washes.

4.10.10 Dip the plate in DI, decant; allow to air dry or dry at 36°± 2°C.

4.10.11 Examine wells with a fluorescent microscope.

4.10.12 A well is considered positive if typical cytoplasmic, apple-green 

fluorescence is observed.

4.10.13 Record the number of wells/dilution showing any characteristic 

fluorescence for each Test Sera and the Reference Serum.

4.11 Calculate the TCID50 of the Test Sera and the virus alone using the SpearmanKärber method as commonly modified.

4.12 The neutralizing ability is determined by subtracting the log of the titer obtained 

with the Test serum from the titer obtained with the virus alone.

Example: Log10 TCID50 Indicator Virus titer 6.0

Log10 TCID50 titer with Test Serum - 2.7

Neutralizing ability = 3.3 logs

5. Interpretation of the Test Results

5.1 No visible contamination or serum toxicity should be observed in any well or any 

dilution of a Test Sera or the Reference Serum.

5.2 The titer of the Reference Serum should be negative for neutralizing antibodies

against BVD.
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5.3 The endpoint titration of the Indicator Virus should fall within 2 standard 

deviations of its mean titer as determined by a minimum of 10 previous titrations.

6. Report of Test Results

Record the neutralizing activity titers for the Test Serum on the test record.

7. References

7.1 Title 9, Code of Federal Regulations, parts 113.215 and 113.216, U.S. 

Government Printing Office, Washington, DC.

7.2 Finney DJ. Statistical Method in Biological Assay. 3

rd ed. 1978. Charles Griffin 

and Co., London.

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

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

8. Summary of Revisions

Version .03

 The Contact information has been updated; however, the Virology Section has elected 

to keep the same next review date for 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 impacting the test were 

made from the previous protocol, the following changes were made to the document:

 The Contact information has been updated.

 Test incubation conditions have been changed from room temperature to 36°± 2°C to 

reflect commonly accepted procedures.
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Appendix I

Minimum Essential Medium (MEM)

MEM (Eagle) with Earles salts q.s. ad 100.0%

Edamin 0.5%

L-Glutamine 1.0%

Gentamicin 50.0 mcg per mL

Penicillin 100.0 units per mL

Streptomycin 100.0 mcg per mL

Amphotericin B 2.5 mcg per mL

Fetal Bovine Serum 10.0%

Appendix II

Phosphate Buffered Saline (PBS-Dulbecco)

NaCl 0.8%

KCl 0.02%

Na2HPO4 0.115%

KH2PO4 0.02%

CaCl2 (anhy) 0.01%

Mgcl2 6H2O 0.01%

Distilled H2O q.s. ad 100.0%