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SAM 112.04

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United States Department of Agriculture

Center for Veterinary Biologics

Testing Protocol

SAM 112

Supplemental Assay Method for Titration of Bovine Viral Diarrhea Virus 

Neutralizing Antibody (Constant Virus - Varying Serum Method)

Date: October 16, 2014

Number: SAM 112.04

Supersedes: SAM 112.03, September 9, 2011

Standard Requirement: 9 CFR 113.215

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

Peg A. Patterson

Approvals: /s/Geetha B. Srinivas Date: 09Dec14

Geetha B. Srinivas, Section Leader

Virology

/s/Byron E. Rippke Date: 10Dec14

Byron E. Rippke, Director

Policy, Evaluation, and Licensing

Center for Veterinary Biologics

/s/Rebecca L.W. Hyde Date: 15Dec14

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 Viral Diarrhea Virus Neutralizing Antibody

(Constant Virus - Varying Serum Method)

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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 Test Serum

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 Viral Diarrhea Virus Neutralizing Antibody

(Constant Virus - Varying Serum Method)

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1. Introduction

This supplemental assay method (SAM) describes an in vitro assay method that determines the 

serum neutralizing (SN) antibody titer to bovine viral diarrhea virus (BVDV) in Test Sera as part 

of the potency requirements for veterinary vaccines. This test applies to BVDV cytopathic strains 

of either genotype 1 or genotype 2. The assay uses Bovine Turbinate (BT) or other permissive

cells and inhibition of viral cytopathic effect as an indicator of the specific SN activity.

Note: For this SAM, the dilution terminology of 1:10, 1:20, etc. specifies 1 part plus 9 parts 

(liquid), 1 part plus 19 parts, etc.

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 Water bath, 37°± 1°C; 56°± 1°C

2.1.3 Pipettors, 50-μL and 500-μL and tips

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

2.1.5 Microscope, inverted light (Model CK, Olympus America Inc.)

2.1.6 Multichannel pipettor, 50- to 300-μL

 x 8- or 12-channel

2.1.7 Ultraviolet light microscope (Model BH2, Olympus America Inc.)

2.2 Reagent/Supplies

Equivalent reagents or supplies may be substituted for any brand name listed below. All 

reagents and supplies must be sterile.

2.2.1 BVDV Reference

2.2.2 BT cells free of extraneous agents as tested by title 9, Code of Federal

Regulations (9 CFR).
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2.2.3 Diluent Medium (National Centers for Animal Health (NCAH) Media 

#20030)

1. 9.61 g minimum essential medium (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 of 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 0.22-μm filter.

7. Aseptically add:

a. 10 mL L-glutamine (200 mM)

b. 50 μg/mL gentamicin sulfate

8. Store at 2°- 7°C.

2.2.4 Growth Medium

1. 900 mL of Diluent Medium

2. Aseptically add 100 mL of gamma-irradiated fetal bovine serum 

(FBS).

3. Store at 2°- 7°C.

2.2.5 Trypsin Versene (TV) Solution (NCAH Media #20005)

1. 8.0 g NaCl

2. 0.40 g KCL

3. 0.58 g NaHCO3

4. 0.50 g irradiated trypsin
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5. 0.20 g versene or disodium salt ethylenediaminetetraacetic acid 

(EDTA)

6. 1.0 g dextrose

7. 0.4 mL 0.5% phenol red

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

9. pH to 7.3 with 7.5% NaHCO3.

10. Filter through a 0.22-μm filter.

11. Store at -20°± 2°C.

2.2.6 Tissue culture plates, 96-well

2.2.7 Polystyrene tubes, 17 x 100-mm

2.2.8 Serological pipette, 10-mL

2.2.9 A negative serum control (NSC)

2.2.10 A positive serum control (PSC) for BVDV, genotype 1

2.2.11 A PSC for BVDV, genotype 2

3. Preparation for the Test

3.1 Personnel qualifications/training

Personnel must have training in cell culture techniques and the propagation and 

maintenance of animal viruses. Personnel shall have an understanding of the 

immunological basis of SN assays and the principles of aseptic techniques.

3.2 Preparation of equipment/instrumentation

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

3.2.2 On the day of test initiation, set a water bath at 36°± 2°C.
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Supplemental Assay Method for Titration of Bovine Viral Diarrhea Virus Neutralizing Antibody

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3.3 Preparation of reagents/control procedures

3.3.1 Preparation of BT Cell Plates

Cells are prepared from healthy, confluent BT cells that are maintained by passing 

every 7 ± 2 days. One day prior to test initiation, cells are removed from the 

growth containers by using TV Solution. Using a multichannel pipettor, add 

200 μL/well of 105 to 105.3cells/mL cells suspended in Growth Medium into all 

wells of a 96-well cell culture plate. 

These become the BT Test Plates. Incubate at 36°± 2°C in a 5 ± 1% CO2

incubator for 24 ± 12 hours. The cell layer should be at least 70% confluent at 24 

hours, and the Growth Medium is not changed unless excess acidity occurs or 

cells are not 70% confluent in 24 hours. Up to 4 Test Sera may be tested per plate.

3.3.2 Preparation of the BVDV Reference Working Dilution

1. On the day of test initiation, a vial of Reference BVDV is rapidly 

thawed in a 36°± 2°C water bath and diluted in Diluent Medium to contain 

50-300 50% tissue culture infectious doses (TCID50) per 25 μL. This 

chosen virus dose is in agreement with the requirements specified in 

9 CFR, Parts 113.215 and 113.311. The procedure for determining the 

Reference BVDV dilution containing 50-300 TCID50 is described in 

Appendix III.

2. Working BVDV back titration

The BVDV Working Dilution is back titrated by preparing serial tenfold 

dilutions (10-1, 10-2, and 10-3) in the following manner.

a. Place 4.5 mL of Diluent Medium into 3 sterile, 17 x 100-mm tubes 

labeled 10-1, 10-2, and 10-3using a 10-mL serological pipette.

b. Using a 500-μL pipettor, transfer 500 μL of BVDV Working 

Dilution to the 10-1tube; mix by vortexing. Discard pipette tip.

c. Using a new pipette tip, transfer 500 μL from the 10-1labeled tube 

to the 10-2tube; mix by vortexing. Discard pipette tip.

d. Repeat Step c to the remaining tube 10-3by transferring 500 μL

from the 10-2dilution and mix the 10-3tube by vortexing.
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3.3.3 Preparation of PSC and NSC

On day of test initiation, serial twofold dilutions of heat-inactivated (56o± 2oC for 

30 ± 5 minutes) PSC and NSC are made from the original whole sera in a 96-well 

tissue culture plate labeled as the Dilution/Transfer Plate (Appendix I). The NSC 

final dilutions range from 1:2 to 1:16 (rows A - D). The range of the PSC twofold 

final dilutions will depend on its SN titer which has been previously determined. 

A sufficient number of dilutions are performed in order to ensure reaching 

an endpoint for the PSC.

1. With a multichannel pipettor, dispense 150 μL of Diluent 

Medium/well into rows B through H.

2. With a 200-μL pipettor, add 150 μL of specific genotype 1 or 2 

undiluted PSC to wells A11 and B11, and 150 μL of undiluted NSC to 

wells A12 and B12 on the Transfer Plate. Use a new tip for each serum 

control.

3. With a multichannel pipettor, mix the samples by aspirating and 

expelling 7 ± 2 times in the wells of row B11 and B12, then transfer 

150 μL of the mixture to the corresponding wells of row C. Discard tips

after use.

4. Using new pipettor tips, repeat Step 3 mixing row C and transferring 

to row D. Continue in this manner for the remaining wells in each column 

changing tips between dilutions until twofold serum dilutions are 

completed. Change tips, mix and discard 150 μL from the highest dilution 

of both the PSC and NSC. 

3.4 Preparation of Test Serum

3.4.1 Test Serum samples are heat inactivated at 56°± 2°C for 30 ± 5 minutes on 

day of test initiation or prior to testing.

3.4.2 On day of test initiation, the Test Sera are diluted in the Dilution/Transfer 

Plate from 1:2 to 1:256 in rows A - H (Appendix I) as described for the PSC and 

NSC in Sections 3.3.3 (Steps 1 through 4).

4. Performance of the Test

4.1 On day of test initiation with new pipette tips, dispense 150 μL of the BVDV 

Working Dilution to each well of the Dilution/Transfer Plate. Mix by gently tapping the 
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sides of the Transfer Plate. The addition of the BVDV Working Dilution to the wells 

results in an additional twofold serum dilution, which becomes the final serum dilution.

4.2 Incubate the Dilution/Transfer Plate at 36°± 2°C for 60 ± 10 minutes in a CO2

incubator.

4.3 Using an 8-channel multipipettor, inoculate 5 wells/dilution/Test Serum with 

50 μL/well of the virus-serum mixture from the Dilution/Transfer plate into the BT Test 

Plate (Appendix II). All 8 dilutions of 1 Test Serum will be inoculated on cells at once. 

Replace tips for each unique Test Serum column being tested. This 96-well plate

becomes the BVDV Test Plate.

4.4 Inoculate 5 wells/dilution with 50 μL/well of the PSC and another set of 5 

wells/dilution of the NSC.

4.5 Inoculate 5 wells/dilution with 25 μL/well of the BVDV Working Dilution and 

Back Titration dilutions.

4.6 Dispense 25 μL of diluent to all wells of the back titration.

4.7 Maintain 5 or more wells as uninoculated cell culture controls per plate.

4.8 Incubate the BVDV Test Plates undisturbed at 36°± 2°C in a CO2 incubator for 5

± 1 days.

4.9 At the end of incubation, read the BVDV Test Plates at 100X magnification on an 

inverted light microscope and examine for BVDV cytopathic effect (CPE).

4.9.1 Results of the Test Serum, PSC, and NSC are recorded as the number of 

CPE negative wells versus total number of wells examined for each dilution of 

that serum.

4.9.2 Results of the BVDV Working Dilution and Back Titration are recorded 

as the number of CPE positive wells versus total number of wells examined for 

each dilution.

4.10 Calculate the endpoint for each Test Serum, PSC, and NSC using the SpearmanKärber method as modified by Finney. The endpoints of the Test Serum, PSC, and NSC 

are reported as SN titer which corresponds to the reciprocal of the highest serum dilution 

that neutralizes the reference BVDV.
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Supplemental Assay Method for Titration of Bovine Viral Diarrhea Virus Neutralizing Antibody

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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

5 = 1 + 5 = 1 + 5 = 0.6 = 2.6

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

antilog of 0.93 = 8.5

Titer of the Test Serum is 1:9

4.11 By the same Spearman-Kärber method, calculate the endpoint of the BVDV Back 

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

25 μL dose.

Example:

100 dilution of the Working BVDV = 5 of 5 wells CPE Positive

10-1dilution of the Working BVDV = 5 of 5 wells CPE Positive

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

10-3dilution of the Working BVDV = 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

5 = 1 + 5 = 1 + 5 = 0.6 = 2.6
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Supplemental Assay Method for Titration of Bovine Viral Diarrhea Virus Neutralizing Antibody

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Titer = (0 - d/2 + [1 • 2.6])= 2.1

antilog of 2.1 = 125.9

Titer of the Working BVDV is 126 TCID50/25 μL dose in the test.

5. Interpretation of the Test Results

5.1 For a valid assay the following criteria must be met; otherwise the test is 

considered a NO TEST and is repeated.

 

5.1.1 The NSC must be negative at the 1:2 dilution.

5.1.2 The endpoint SN titer of the PSC should vary by no more than twofold 

from its previous determined mean titer as established from a minimum of 10 SN 

titrations.

5.1.3 The uninoculated cell controls cannot exhibit any CPE or cloudy media 

that would indicate contamination.

5.1.4 The BVDV Back Titration titer must be between 100-300 TCID50/25 μL

(Section 4.11).

5.2 For a SATISFACTORY TEST, the postvaccination SN titers shall meet the 

requirements as stated in an Animal and Plant Inspection Service (APHIS) approved

Outline of Production.

5.3 If postvaccination SN titers are less than the requirements in an APHIS approved

Outline of Production, the sera will be retested (first retest).

5.3.1 If the SN titers of the Test Serum from the first valid retest are less than 

the required titers in an APHIS approved Outline of Production, the serial is 

UNSATISFACTORY.

5.3.2 If the SN titers of the Test Serum from the first valid retest are greater than 

or equal to the titer in an APHIS approved Outline of Production, the serum will 

be retested (second retest).

5.3.3 If the SN titers of the Test Serum from the second valid retest are greater 

than or equal to the titer in an APHIS approved Outline of Production, the serial is 

SATISFACTORY.
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5.3.4 If the SN titers of the Test Serum from the second valid retest are less than 

the titers in an APHIS approved Outline of Production, the serial is 

UNSATISFACTORY.

6. Report of Test Results

6.1 Results are reported as SN titers.

6.2 Record all test results on the test record.

7. References

7.1 Title 9, Code of Federal Regulations, part 113.6, U.S. Government Printing 

Office, Washington, DC.

7.2 Finney, DJ 1978. Statistical method in biological assay. Griffin, London. 3rd 

edition, pp. 394-401.

7.3 Cottral, GE, (Ed.) 1978, Manual of standard methods for veterinary 

microbiology. Comstock Publishing Associates, Ithaca, NY, pg. 731.

8. Summary of Revisions

Version .04

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

to keep the same next review date for the document.

Version .03

 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 .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:

 The document number has been changed from MVSAM0112 to SAM 112.
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Supplemental Assay Method for Titration of Bovine Viral Diarrhea Virus Neutralizing Antibody

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 The Contact information has been updated.

 References to embryonic bovine kidney primary (EBKp) cells have been changed to 

Bovine Turbinate (BT) cells and the fluorescence antibody (FA or IFA) reference has 

been removed.

 2.2: Phosphate Buffered Saline; Monoclonal antibody to BVDV type 1 or 2; and 

Anti-Reference Virus Fluorescein Isothiocyanate Labeled Conjugates have been 

removed from the document. The use of a 500-mL plastic wash bottle and acetone 

has also been removed from the document.

 2.2.3: The formulation for the Diluent Medium has been changed and penicillin, 

streptomycin and amphotericin B have been removed.

 3.3.2: Reference to the 9 CFR, Parts 113.215 and 113.311 has been added for 

clarification of the procedure.

 4.12: The processes of fluorescent staining of plate cultures have been deleted.

 Virus titrations and dilutions calculations have been added to the Appendices.

 The recommended cell culture of EBKp has been changed to BT 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 Amega system.
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Appendix I

Dilution/Transfer Plate

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

A TS1

dilute

1:2

TS2

dilute

TS3

dilute

TS4

dilute

TS5

dilute

TS6

dilute

TS7

dilute

TS8

dilute

TS9

dilute

TS 10

dilute

TS 11

dilute

TS 12

dilute

B 

4

C 

8

D 

 16

E 

32

F 

64

G 

128

H 

256

TS= Test Serum
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Appendix II

BVDV Test Plate

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

A 

1:2

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

B 

4

C 

8

D 16

E 32

F 64

G 

128

H 

256

TS= Test Serum

CC= Cell Control

BVDV Control Serum Plate

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

A 

1:2

PSC PSC PSC PSC PSC CC PSC PSC PSC PSC PSC CC

B 

4

C 

8

D 16

E 32

F 64

G 

128

H 

256
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Appendix III

Titration of BVDV and Determination of the Dilution Containing the Optimal Virus Dose 

for the Beta SN Test

1. Label eight 17 x 100-mm plastic tubes by identifying one tube as 10-1, one 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 36oC 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 BT cell plate as 

prepared in 3.3.1. The plate is then incubated in a 36o± 2oC, 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 BVDV virus 

titration. The titer is expressed as log10 tissue culture infective doses fifty 

(TCID50) per 25 μL dose.

Example:

10-1dilution of the Working BVDV = 5 of 5 wells CPE Positive

10-2dilution of the Working BVDV = 5 of 5 wells CPE Positive

10-3dilution of the Working BVDV = 5 of 5 wells CPE Positive

10-4dilution of the Working BVDV = 5 of 5 wells CPE Positive

10-5dilution of the Working BVDV = 3 of 5 wells CPE Positive 10-6dilution of the 

Working BVDV = 0 of 5 wells CPE Positive
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Titer = (X - d/2 + [d • S]) where:

X = Log10 of lowest dilution (=1)

d = Log10 of dilution factor (=1)

S = Sum of proportion of CPE Positive

5 5 5 5 3

5 = 1 + 5 = 1 + 5 = 1 + 5 = 1 + 5 = 0.6 = 4.6

Titer = (1 - 1/2 + [1 • 4.6]) = 5.1

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 BVDV 

dilution must be confirmed by performing a back titration. A series of three to five 

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.