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SAM 126.04

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United States Department of Agriculture

Center for Veterinary Biologics

Testing Protocol

SAM 126

Supplemental Assay Method for Titration of Bovine Rotavirus Antibody 

(Constant Virus-Varying Serum Method)

Date: October 23, 2014 Approved, Pending Standard Requirement

Number: SAM 126.04

Supersedes: SAM 126.03, February 9, 2011

Standard Requirement: 

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

Peg A. Patterson

Approvals: /s/Geetha B. Srinivas Date: 11Dec14

Geetha B. Srinivas, Section Leader

Virology

/s/Byron E. Rippke Date: 16Dec14

Byron E. Rippke, Director

Policy, Evaluation, and Licensing

Center for Veterinary Biologics

/s/Rebecca L.W. Hyde Date: 16Dec14

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 Rotavirus 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 Rotavirus Antibody 

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1. Introduction

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

(SN) antibody titer against bovine rotavirus (BRota). The assay uses a constant amount of virus 

to test varying dilutions of serum for antibodies against Group A bovine rotavirus in a cell 

culture system.

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 Water bath, 37°± 1°C; 56°± 1°C

2.1.3 Pipettors, 200-μL and 500-μL and tips

2.1.4 Vortex mixer

2.1.5 Microscope, inverted light

2.1.6 Multichannel pipettor, 50- to 300-μL

 x 8- or 12-channel

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 Cell Cultures

Multiple 96-well disposable plates are seeded (0.2 mL/well) with rhesus monkey 

kidney (MA-104) cells. Cells must be free of extraneous agents. The cells are 

seeded at a density that will produce 90%-100% confluency after 2 days of 

incubation.
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2.2.2 Growth Medium

The cells are grown in Minimum Essential Medium (MEM) with 7% fetal bovine 

serum and additives (Appendix I) at a temperature of 35°- 37°C in an incubator 

containing an atmosphere of 5% carbon dioxide (CO2) and a relative humidity of 

70% to 80%. Growth Medium is not changed unless excess acidity occurs or cells 

are not growing well.

2.2.3 Maintenance Medium

Maintenance Medium (Appendix II) without serum is used to rinse the cells prior 

to inoculation. It is also used as a diluent, in the presence of pancreatin, for the 

serum-virus neutralization assay.

2.2.4 Indicator Virus

The National Veterinary Services Laboratories (NVSL) reference bovine 

rotaviruses for serotypes 6 (NCDV-Lincoln strain) and 10 (B223 strain) are used 

as controls for the cell system.

2.2.5 Primary Antibody

When indirect immunofluorescence (IFA) and not cytopathic effect (CPE) is used 

to titer the virus, serotype- or strain-specific antisera or monoclonal antibodies are 

used as primary antibody.

2.2.6 Fluorescent Antibody Conjugate

NVSL reference fluorescein isothiocyanate-conjugated immunoglobulin-specific 

antiserum is used in the IFA assay.

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.
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3.2.2 On the day of test initiation, set a water bath at 37°± 1°C.

3.3 Preparation of reagents/control procedures

3.3.1 Preparation of MA-104 Plate

Cells are prepared from healthy, confluent MA-104 cells that are maintained by 

passing every 5 ± 2 days. Two days prior to test initiation, cells are removed from 

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

200 μL/well of 105.4 to 105.6 cells/mL cells suspended in Growth Medium into all 

wells of a 96-well cell culture plate. Prepare 1 MA-104 plate for the controls and 

BRota Positive Control Working Dilution. Each additional plate allows testing of 

2 Test Serials either pre- or postvaccination. These become the MA-104 Test 

Plates. Incubate at 36°± 2°C in a CO2 incubator for 2 days ± 12 hours. Growth 

Medium is not changed unless excess acidity occurs or cells are not confluent in 2 

days.

3.3.2 Preparation of the BRota Positive Control Working Dilution

1. On the day of test initiation, a vial of BRota Positive Control 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 50 μL. Prior 

testing of the virus is used to determine the dilution needed to obtain 50 to 

300 TCID50 for testing. This becomes the BRota Working Dilution.

2. The BRota Working Dilution is back titrated by preparing serial 

tenfold dilutions (10-1, 10-2, and 10-3) and allowing them to remain at room 

temperature (23°± 2°C).

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

labeled 10-1-10-3using a 10-mL serological pipette.

b. Using a 500-μL pipettor, transfer 500 μL of BRota 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 2.c to the remaining tube, transferring 500 μL from 

the previous dilution.
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3.4 Preparation of the sample

3.4.1 On day of test initiation or prior to testing (inactivation of pre- and 

postserums maybe done at time of collection), serums are heat-inactivated at 

56°± 2°C for 30 ± 5 minutes.

3.4.2 On day of test initiation, the Test Serums (pre- and/or postvaccination 

serums) are diluted in the Transfer Plate from 1:2 to 1:256 in rows A-H 

(Appendix I) of a 96-well tissue culture plate labeled as the Test Serum Transfer 

Plate as described for the PSC and NSC.

4. Performance of the Test

4.1 The Indicator Virus is diluted to contain 100 to 350 TCID50 per 0.2 mL, using 

Maintenance Media containing a previously titrated amount of pancreatin, the maximum 

that the MA-104 cells will tolerate. This dilution is determined by previous titrations and 

is designated the “Stock Virus.” The dilution factor is calculated by dividing the titer of 

the Indicator Virus by the desired titer of Stock Virus.

4.2 The serum to be assayed is heat-inactivated at 56°C for 30 minutes. Serial twofold 

dilutions are made in sterile tubes with diluent containing pancreatin and mixed using a 

Vortex or similar type of mixer.

4.3 An equal volume of Stock Virus and of each serum dilution are added together, 

mixed, and allowed to incubate at 37°C for 60 minutes. The mixing of equal volumes of 

serum and virus results in a further twofold dilution of serum.

4.4 Cells that have been seeded in 96-well plates 2 to 3 days previously are inverted 

and the Growth Medium removed by gentle shaking and tamping on sterile gauze. The 

cells are rinsed with 0.2 mL of Maintenance Medium per well, the medium again 

decanted from the plate, and the cells refed with 0.2 mL of Maintenance Media which 

remains on the plate for 1 hour at 37°C.

4.5 The final rinse is removed from the cells, as above. Each well is inoculated with 

0.2 mL per well of each Stock Virus-Serum Dilution mixture, at a minimum of 4 wells 

per dilution. A minimum of 8 wells remain uninoculated with virus, to serve as negative 

cell controls; they receive 0.2 mL per well of only the pancreatin-containing diluent.

4.6 The Stock Virus is back-titrated by preparing serial tenfold dilutions (100, 10-1, 

10-2, 10-3, 10-4) in diluent containing pancreatin. These are then mixed with an equal 

volume of diluent containing pancreatin and at least 4 wells per dilution are inoculated 

with 0.2 mL of the mixture.
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4.7 The plates are incubated at 37°C, in an atmosphere of 5.0% CO2 and high 

humidity, for 5 days. After 5 days, the cells can be examined for CPE typical of bovine 

rotavirus and the 50% endpoints calculated. The TCID50 of the Stock Virus is calculated 

by the Spearman-Kärber method; its titer must be between 50 and 350 TCID50 per 0.2 mL

for a test to be valid. The cells in the Negative Control wells must remain normal.

4.8 Certain strains of bovine rotavirus may not exhibit pronounced CPE, thus an IFA

assay may be necessary to determine their titer.

4.8.1 After the medium is decanted, the cells are gently rinsed in phosphate 

buffered saline (PBS), then in deionized water (DI). The cells are fixed in a 

solution of 80% acetone-20% DI at 4°C for 15 minutes. The acetone is discarded 

and the plates air-dried.

4.8.2 The wells are covered with 0.05 mL per well of previously-titrated 

dilution of specific primary antibody and held in a high humidity, 37°C incubator 

for 30 minutes. Excess primary antibody is washed from the plates by two gentle 

PBS rinses and one DI rinse. The plates are shaken gently and lightly touched to 

an absorbent towel to remove excess moisture.

4.8.3 While still moist, wells are covered with 0.05 mL per well of speciesspecific, conjugated anti-immunoglobulin antiserum. Again, the plates are 

incubated at 37°C for 30 minutes. Washing is repeated, as in Step 2. The plates 

are air-dried, face-down.

4.8.4 The cell monolayers are examined by fluorescent microscopy using a 

Ploem illuminator and blue light (Xenon lamp). Any cells showing 

immunofluorescence characteristic of bovine rotavirus are considered positive and 

the 50% endpoints calculated. The TCID50 of the Stock Virus is calculated using 

the Spearman-Kärber method; its titer must be between 50 and 350 TCID50 per 

0.2 mL for a test to be valid. Also, the non-inoculated wells must be negative for 

immunofluorescence.

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 without prejudice.

5.1.1 The NSC must have an SN titer of < 1:2.
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5.1.2 The SN titer of the PSC should vary by no more than twofold from its 

mean titer as established from a minimum of 10 previously determined SN 

titrations.

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

that would indicate contamination.

5.1.4 The BRota Back Titration titer must be between 50 to 350 TCID50/25 μL. 

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

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

of Production.

5.3 If postvaccination titers are less than the requirements in an APHIS filed Outline 

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

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

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

UNSATISFACTORY.

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

equal to the titer in an APHIS filed Outline of Production, the serum will be 

retested (second retest).

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

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

SATISFACTORY.

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

titers in an APHIS filed 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.
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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, pg. 508.

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

microbiology. Comstock Publishing Associates, Ithaca, New York, 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 Contact information has been updated.

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:

 Contacts, Joseph Hermann and Peg Patterson, have been added to the document.
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Appendix I

Growth Medium

MEM (Eagle) with Earles salts 1.0 packet

Deionized water q.s. 1.0 liter

Sodium bicarbonate 2.2 gram

Gentamicin sulfate 50.0 mg

Penicillin 25,000 U

Streptomycin 100.0 mg

Heat inactivated or irradiated fetal bovine serum 70.0 mL

200 mM L-Glutamine (100X) 292.0 mg

0.22 micron filtration

Appendix II

Maintenance Medium

MEM (Eagle) with Earles salts 1.0 packet

Deionized water q.s. 1.0 liter

Sodium bicarbonate 2.2 gram

Gentamicin sulfate 50.0 mg

Amphotericin B 5.0 mg

Penicillin 100,000 U

Streptomycin 100.0 mg

200 mM L-Glutamine (100X) 292.0 mg

0.22 micron filtration