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SAM 120.03

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United States Department of Agriculture

Center for Veterinary Biologics

Testing Protocol

SAM 120

Supplemental Assay Method for the In vitro Potency Assay of Bovine 

Respiratory Viruses in Killed Vaccines

Date: October 16, 2014

Number: SAM 120.03

Supersedes: SAM 120.02, February 9, 2011

Standard Requirement: 9 CFR 113.8

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

Peg A. Patterson

Approvals:

/s/Geetha B. Srinivas Date: 12Nov14

Geetha B. Srinivas, Section Leader

Virology

/s/Byron E. Rippke Date: 24Nov14

Byron E. Rippke, Director

Policy, Evaluation, and Licensing

Center for Veterinary Biologics

/s/Rebecca L.W. Hyde Date: 25Nov14

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 In vitro Potency Assay of Bovine Respiratory Viruses in Killed Vaccines

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Table of Contents

1. Introduction

2. Materials

3. Performance of the Test

4. Interpretation of the Test Results

5. Report of Test Results

6. References

7. Summary of Revisions
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Supplemental Assay Method for the In vitro Potency Assay of Bovine Respiratory Viruses in Killed Vaccines

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1. Introduction

This Supplemental Assay Method (SAM) describes an in vitro assay for determining the potency 

of killed vaccines containing Bovine Rhinotracheitis (BRV), Bovine Virus Diarrhea (BVD), 

Bovine Parainfluenza-3 (PI3), and Bovine Respiratory Syncytial Viruses (BRSV) relative to a 

reference vaccine.

2. Materials

2.1 96-well microtiter plate

2.2 Coating buffer: 0.05 M sodium carbonate/bicarbonate pH 9.6. Store at 4°C; use 

within 5 days.

2.2.1 1.59 g Na2CO3

2.2.2 2.93 g NaHCO3

2.2.3 Distilled H2O, q.s. to 1 L

2.2.4 Adjust pH to 9.6 ± 0.1.

2.3 Anti-viral agent capture antibody, using 1 capture antibody only, and assay 1 viral 

fraction in each test. Reference quantities are available from the National Veterinary 

Services Laboratories (NVSL). Dilute in coating buffer according to instructions.

2.3.1 BRV antibody

2.3.2 BVD antibody

2.3.3 Bovine PI3 antibody

2.3.4 BRSV antibody

2.4 Blocking buffer: 1% casein in coating buffer. Store at 4°C; use within 5 days.

2.4.1 1 g casein

2.4.2 100 mL coating buffer

2.4.3 Heat 2 to 3 minutes until boiling in a microwave on high setting.
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2.5 0.01 M PBS. Store at 4°C.

2.5.1 1.33 g Na2HPO4

2.5.2 0.22 g Na2H2PO4•H2O

2.5.3 8.5 g NaCl

2.5.4 Distilled H2O, q.s. to 1 L

2.5.5 Autoclave 15 minutes at 121°C.

2.6 Diluent buffer: 1% casein in 0.01 M PBS. Prepare fresh each time test is 

performed.

2.6.1 1 g casein

2.6.2 100 mL 0.01 M PBS

2.6.3 Heat 2 to 3 minutes until boiling in a microwave on high setting.

2.7 Wash reagent: 0.05% Tween-20 in 0.01 M PBS

2.8 Secondary antibody: Available from the NVSL unless noted. Dilute in dilutent 

buffer according to instructions.

2.8.1 BRV monoclonal antibody (MAb):

1. 1B8

2. 2H6

2.8.2 BVD biotin-labeled polyclonal antibody

2.8.3 PI3 MAb:

1. 240-12D

2. 260-10B

2.8.4 BRSV MAb 8G12
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2.9 Conjugate

2.9.1 Horseradish peroxidase-labeled goat anti-mouse IgG, diluted 1:3000 in 

0.01 M PBS, is to be used with the BRV, PI3, and BRSV MAbs.

2.9.2 Peroxidase conjugated streptavidin, diluted 1:4000 in 0.01 M PBS, is to be 

used with biotin-labeled polyclonal BVD antibody.

2.10 Substrate: 2,2’-azino-di (3-ethylbenzthiazoline sulfonic acid) (ABTS)

Mix equal parts of Solution A and Solution B as supplied just before use in the in vitro

test.

2.11 Reference vaccine(s) to be supplied by the licensee. The reference vaccine(s) 

should have the same dose size and composition as the test serial, be validated in a host 

immunogenicity study and shown to work in a parallel line assay. Refer to the Guidelines 

for measuring the relative potency of veterinary biologics by ELISA, dated April 26, 

1991.

3. Performance of the Test

If the test serial vaccine is treated to release antigen from adjuvant, it shall be a method 

acceptable to the Animal and Plant Health Inspection Service (APHIS) and shall be applied to 

the reference vaccine as well.

3.1 Coat microtiter plate with 100 μL/well of capture antibody diluted in coating 

buffer. Cover with sealing tape. Incubated overnight at 4°C.

3.2 Decant capture antibody. Blot inverted plate on paper towels. Add 100 μL/well of 

blocking buffer. Incubate 1 hour at room temperature.

3.3 Rinse microtiter plate 3 times using 200 μL/well of wash reagent. Remove 

residual wash reagent by blotting inverted plate on paper towels after 3rd wash. Prevent 

drying of plate between reagent additions.

3.4 Prepare serial twofold dilutions of the reference and test serial vaccines in a 

separate dilution plate.

3.4.1 Add 150 μL/well of diluent buffer to rows B-H of a 96-well plate.

3.4.2 Add 300 μL/well of reference vaccine to A1 through A3 of the dilution 

plate. Add 300 μL/well of the test serial vaccine to A4 through A6 of the dilution 
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plate. Additional test serials may be tested in A7 through A9 and A10 through 

A12.

3.4.3 Using a multichannel pipette, transfer 150 μL of row A to row B. 

Thoroughly mix contents of wells in row B. Transfer 150 μL to the corresponding 

wells in row C. 

Note: One set of pipette tips may be used for diluting the reference and serial 

vaccines.

3.4.4 Thoroughly mix contents of wells in row C. Transfer 150 μL to the 

corresponding wells in row D.

3.4.5 Continue serial twofold dilutions through row G. After thoroughly mixing 

the contents in wells G, discard 150 μL from row G. The dilutions of the vaccines 

range from undiluted to 1:64.

Note: Other dilution schemes for the reference vaccine and test serial may be 

used to obtain optimal optical density (OD) readings.

3.4.6 Row H is the blanking row.

3.5 Use the multichannel pipette to transfer 100 μL aliquots from row H of the 

dilution plate to the corresponding row H of the capture antibody coated plate. Continue 

transfer of all rows of the dilution plate from row G through row A. One set of pipette 

tips may be used to transfer. Incubate 1 hour at 37°C on a microtiter plate shaker.

3.6 Rinse plate again as in Step 3.3.

3.7 Add 100 μL/well of secondary antibody diluted in diluent buffer to all wells of 

the plate. Incubate 1 hour at 37°C.

3.8 Rinse plate again as in Step 3.3.

3.9 Add 100 μL/well of conjugate diluted in 0.01 M PBS. Incubate 30 minutes at 

37°C.

3.10 Rinse plate 3 times with 0.01 M PBS without Tween-20. Remove residual PBS by 

blotting inverted plate on paper towels after the 3rd wash.

3.11 Add 100 μL/well of ABTS. Incubate 45 minutes at room temperature.
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3.12 Read plate on an ELISA reader with 405 nm for the test filter and 490 nm for the 

reference filter.

3.13 Blank reader on well(s) containing all reagents except the fraction being tested 

(row H). If reader blanking is not done, the mean OD value obtained from the blank(s) 

should be subtracted from all other OD values before any data analysis.

4. Interpretation of the Test Results

A test that results in no valid lines is considered a no test and may be repeated. The reported 

relative potency (RP) of the test serial is the highest RP from the top 3 scores calculated using 

SoftMax Pro 6.3 GxP. Test serials with RP ≥ 1.0 are considered satisfactory. Retest procedures 

are covered under title 9, Code of Federal Regulations (9 CFR), part 113.8. 

Test serials with a RP < 1.0 may be retested up to a total of 3 times. At least 50% of all valid 

tests must be satisfactory for the test serial to be considered satisfactory. If the test serial is not 

retested, the serial is considered unsatisfactory.

5. Report of Test Results

Record all test results on the test record.

6. References

Title 9, Code of Federal Regulations, part 113.8, U.S. Printing Office, Washington, DC.

7. 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 
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the outcome of the test, the following changes were made to the document:

 The Contact information has been updated.