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SAM 106.03

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United States Department of Agriculture

Center for Veterinary Biologics

Testing Protocol

SAM 106

Supplemental Assay Method for Titration of Neutralizing Antibody (Constant 

Virus-Varying Serum Method)

Date: October 16, 2014 Approved, Pending Standard Requirement

Number: SAM 106.03

Supersedes: SAM 106.02, February 9, 2011

Standard Requirement: 

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

Peggy A. Patterson

Approvals: /s/Geetha B. Srinivas Date: 31Dec14

Geetha B. Srinivas, Section Leader

Virology

/s/Byron E. Rippke Date: 06Jan15 

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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Table of Contents

1. Introduction

2. Materials

2.1 Cell cultures

2.2 Indicator viruses

2.3 Diluent

2.4 Test serums

2.5 Conjugate

2.6 Guinea pig red blood cells (RBCs) for the hemadsorption (HAd) test

3. Preparation for the Test

3.1 Personnel qualifications/training

3.2 Preparation of reagents/control procedures

3.3 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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1. Introduction

This Supplemental Assay Method (SAM) describes an in vitro assay method which employs a 

cell culture system for determining the antibody titer of serum against Bovine Rhinotracheitis 

(IBR), Bovine Virus Diarrhea (BVD), and Parainfluenza-3 (PI3) Viruses.

2. Materials

2.1 Cell cultures

Roller tubes (16 x 150-mm) containing monolayers of primary bovine embryonic kidney 

(BEK) cells are used for IBR and PI3 serum neutralization (SN) tests, and Leighton tubes 

containing monolayer BEK cells on coverslips (10.5 x 35-mm) are used for BVD SN 

tests. Cells found free from extraneous agents are used in these tests.

2.1.1 Primary BEK cells are grown from trypsinized kidney cortical tissue, 

frozen and stored at -80°C, and tested for extraneous agents.

2.1.2 Frozen cells are thawed, suspended in growth medium (Appendix I), and 

1 mL amounts dispensed into Leighton or roller tubes.

2.1.3 The tubes containing the cells are incubated in stationary racks at 

36°- 37°C until the monolayer is at least 80% confluent. The growth medium is 

replaced with maintenance medium (Appendix II) just before the tubes are 

inoculated.

2.2 Indicator viruses

Reference IBR, BVD, or PI3 viruses are used.

2.3 Diluent

Maintenance medium, without serum, is used to make dilutions of the virus and serum.

2.4 Test serums

2.4.1 Serums to be tested

2.4.2 Negative control serum
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2.5 Conjugate

Conjugated BVD specific immune serum is used in the BVD SN test system.

2.6 Guinea pig red blood cells (RBCs) for the hemadsorption (HAd) test

2.6.1 Blood from healthy guinea pigs is collected aseptically in an equal volume 

of sterile Alsever’s solution (Appendix III).

2.6.2 The RBCs are washed 3 times in Alsever’s solution and sedimented each 

time by centrifugation at 1,000 rpm (250 Gs) for 15 minutes.

2.6.3 The RBCs are stored at 5°C as a 50% suspension in Alsever’s solution.

2.6.4 For the hemadsorption test, the RBCs are diluted to a 0.5% suspension in 

phosphate buffered saline (PBS) (Appendix IV).

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.

3.2 Preparation of reagents/control procedures

Dilution of indicator virus: The indicator virus is diluted to contain 100 to 500 

TCID50/0.1 mL; 0.1 mL is inoculated into each tissue culture tube in the test system. This 

dilution is determined by previous titrations and is designated as the “stock virus.”

Calculation of the dilution factor is as follows: Divide the titer of the indicator virus by 

the desired titer of stock virus. This equals the dilution factor.

Example:

 Indicator virus titer = dilution factor

Stock virus titer wanted

1,000,000 TCID50/0.1 mL = 5,000

 200 TCID50/0.1 mL

The indicator virus is diluted 1:5,000.
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3.3 Preparation of the sample

Dilution of test serum: The serum is heat-inactivated at 56°C for 30 minutes. Serial 

twofold dilutions are made in sterile tubes containing diluent. Transfers are made with a 

1-mL pipette and mixing is done by using a mixer (Vortex or similar type).

Twofold dilutions are made as follows:

3.3.1 One mL diluent is added to tubes 2, 3, 4, and 5.

3.3.2 One mL serum is added to tubes 1 and 2. Pipette is discarded and tube 2 is 

mixed. Tube 1 contains 1 mL of undiluted serum. Tube 2 contains a 1:2 dilution 

of the serum.

3.3.3 One mL is transferred from tube 2 to tube 3. Pipette is discarded and tube 

3 is mixed. Tube 3 contains a 1:4 dilution of the serum.

3.3.4 This process is continued until the desired number of serum dilutions are 

made. One mL from the last serum dilution tube is discarded.

4. Performance of the Test

4.1 Serum neutralization of virus

An equal volume of stock virus (1 mL) is added to each serum dilution tube (1 mL), 

mixed, and allowed to incubate at room temperature for 45 minutes. Each of 5 cell 

culture tubes is inoculated with 0.2 mL of the serum-virus mixture. The mixing of equal 

volumes of serum and virus results in a further twofold dilution of serum. Thus, the 

undiluted serum (tube 1) becomes a 1:2 final dilution, the initial 1:2 becomes 1:4, etc.

4.2 Controls

4.2.1 The stock virus is titrated by preparing serial tenfold dilutions (100, 10-1, 

10-2, 10-3, and 10-4) and allowing them to remain at room temperature along with 

the serum-virus mixtures. Five cell culture tubes are inoculated with 0.1 mL of 

each virus dilution.

4.2.2 A known negative serum control is tested at a 1:2 dilution along with the 

test serums.

4.2.3 Five uninoculated cell culture tubes are incubated and processed along 

with the other cultures as a check on the test system.
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5. Interpretation of the Test Results

The 50% endpoints of the serum and virus are calculated by the method of Reed and Muench or 

Spearman-Kärber. The neutralization dose50 of serum is based on the dilution showing complete 

neutralization of the test dose of virus.

The stock virus 50% endpoint must be between 100 and 500 TCID50/0.1 mL for a test to be 

valid. The negative serum control must not neutralize virus. The cells in the uninoculated control 

tubes must remain normal.

6. Report of Test Results

6.1 Bovine Rhinotracheitis

The inoculated BEK roller tubes are incubated at 35°- 37°C for 4 to 6 days. The tubes are 

examined for cytopathic effect (CPE) typical of IBR virus. The number of tubes found 

positive and negative for CPE is recorded and the 50% endpoints calculated.

6.2 Bovine Virus Diarrhea

The inoculated Leighton tubes are incubated at 35°- 37°C for 4 to 6 days. The coverslips 

are removed from the tubes and processed for reading by the fluorescent antibody (FA) 

technique. The cells on the coverslips are stained as follows:

6.2.1 Coverslips are removed from the tubes and placed in racks.

6.2.2 They are rinsed in PBS, then in distilled water (DW), and dried.

6.2.3 They are fixed in cold acetone for 15 minutes, then dried thoroughly.

6.2.4 The cells are covered with conjugated BVD-specific immune serum and 

held in a high-humidity incubator at 37°C for 30 minutes.

6.2.5 Conjugate is drained and the coverslips are washed in a gently circulating 

PBS bath for 10 minutes, rinsed in DW, and dried.

6.2.6 Coverslips are mounted on glass slides with the cells down using FA 

mounting fluid.

Monolayer cells are examined by fluorescence microscopy with dry darkfield condenser. 

The number of slides positive and negative for fluorescence is recorded and the 50% 

endpoints calculated.
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6.3 Parainfluenza-3

The inoculated BEK roller tubes are incubated at 35°- 37°C for 4 to 6 days. The cell 

layers are examined by one or both of the following methods:

6.3.1 Cytopathic effect

The tubes are examined for CPE typical of PI3 virus. The number of tubes found 

positive and negative for CPE is recorded and the 50% endpoints calculated.

6.3.2 Hemadsorption test

1. Fluids are poured from the tubes.

2. The cells are washed once with PBS.

3. To each tube is added 1 mL of a 0.5% suspension of RBCs.

4. The tubes are placed so that the cell monolayer is covered with the 

RBC suspension and allowed to stand 15 to 20 minutes at room 

temperature.

5. The suspension of RBCs is poured off and the monolayers are washed 

3 times with PBS.

6. The PBS is drained from the tubes and the monolayers are examined 

microscopically from hemadsorption.

The number of tubes positive and negative for HAd is recorded and the 

50% endpoints calculated.

7. References

Title 9, Code of Federal Regulations, U.S. Government Printing Office, Washington, DC.
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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 were made that impact 

the outcome of the test, the following changes were made to the document:

 The Contact information has been updated.
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Appendices

Appendix I

Growth Medium

Lactalbumin hydrolysate 0.5%

Hanks BSS q.s. ad 100.0%

Antibiotics 

Penicillin 100 units/mL

Streptomycin 100 mcg/mL

Kanamycin 160 mcg/mL

Amphotericin B 2 mcg/mL

Ten percent fetal calf serum is added.

Appendix II

Maintenance Medium

Lactalbumin hydrolysate 0.5%

MEM (Eagle) q.s. ad 100.0%

Antibiotics

Penicillin 100 units/mL

Streptomycin 100 mcg/mL

Kanamycin 160 mcg/mL

Amphotericin B 2 mcg/mL

Two percent fetal calf serum is added.

Appendix III

Alsever’s Solution

Dextrose 2.05%

Sodium citrate 0.8%

Sodium chloride 0.42%

Citric acid 0.55%

Distilled H2O q.s. ad 100%
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Appendix IV

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%