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SAM 113.05

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United States Department of Agriculture

Center for Veterinary Biologics

Testing Protocol

SAM 113

Supplemental Assay Method for Titration of Parainfluenza 3 Neutralizing 

Antibody (Constant Virus - Varying Serum Method)

Date: May 12, 2017

Number: SAM 113.05

Supersedes: SAM 113.04, October 16, 2014

Standard Requirement: 9 CFR 113.200

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

Peg A. Patterson.

Approvals: /s/Geetha B. Srinivas Date: 12Jul17

Geetha B. Srinivas, Section Leader

Virology

/s/Paul J. Hauer Date: 13Jul17

Paul J. Hauer, Director

Policy, Evaluation, and Licensing

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.

Entered into CVB Quality Management System

by: /s/Linda S. Snavely 13Jul17 

 Linda S. Snavely Date

 Quality Management Program Assistant
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Supplemental Assay Method for Titration of Parainfluenza 3 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 Parainfluenza 3 Neutralizing Antibody

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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 Parainfluenza 3 virus (PI3V) in Test Sera as part of the 

potency requirements for veterinary vaccines. The assay uses Madin-Darby bovine kidney 

(MDBK) cells and inhibition of viral cytopathic effect (CPE) as an indicator of the specific SN 

activity.

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

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

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 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 PI3V Positive Control

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

Regulations (9 CFR).

2.2.3 Diluent Medium (National Centers for Animal Health (NCAH) Media

#20030)
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1. 9.61 g minimum essential medium with Earles salts without 

bicarbonate

2. 2.2 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 Section 2.2.4(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 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 Diluent Medium

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

(FBS).

3. Store at 4°± 2°C.

2.2.5 Maintenance Medium

1. 980 mL of Diluent Medium

2. Aseptically add 20 mL of gamma-irradiated FBS.

3. Store at 4°± 2°C.

2.2.6 Alsever’s Solution (NCAH Media #20031)

1. 20.5 g dextrose (C6H12O6)

2. 8.0 g sodium citrate (Na3C6H5O72H2)
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3. 4.2 g sodium chloride (NaCl)

4. 0.55 g citric acid (C6H8O7)

5. Q.S. to 100 mL with DI.

6. Filter through a 0.22-μm filter.

7. Store at 4°± 2°C.

2.2.7 Phosphate buffered saline 10X (PBS) (NCAH Media #30069)

1. 8.0 g NaCl

2. 0.2 g potassium chloride (KCl)

3. 0.2 g potassium phosphate, monobasic, anhydrous (KH2PO4)

4. 1.15 g sodium phosphate dibasic, anhydrous (Na2HPO4) 

5. Q.S. to 1000 mL with DI, adjust the pH to 7.0-7.3 with 5N sodium 

hydroxide (NaOH) and autoclave at 15 psi, 121°± 2°C for 35 ± 5 minutes.

6. Store at 4°± 2°C.

2.2.8 1X PBS

1. 100 mL 10X PBS

2. 900 mL DI

3. Store at 4°± 2°C

2.2.9 Guinea pig red blood cells (RBCs) in an equal volume of Alsever’s 

Solution

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

1. 8.0 g NaCl

2. 0.40 g KCL

3. 0.58 g NaHCO3
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4. 0.50 g irradiated trypsin

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 NaHCO3

10. Filter through a 0.22-μm filter.

11. Store at -20°± 4°C.

2.2.11 Tissue culture plates, 96-well

2.2.12 Polystyrene tubes, 17 x 100-mm

2.2.13 Conical tube, 50-mL

2.2.14 Serological pipette, 10-mL

2.2.15 PI3V inactivated negative serum control (NSC)

2.2.16 PI3V inactivated positive serum control (PSC)

2.2.17 Plastic wash bottle, 500-mL

3. Preparation for the Test

3.1 Personnel qualifications/training

Personnel must have training in cell culture technique 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 technique.

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 MDBK Plate

Cells are prepared from healthy, confluent MDBK 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 MDBK plate for the controls and 

PI3V Positive Control Working Dilution. Each additional plate allows testing of 2 

Test Serials either pre- or postvaccinations. These become the MDBK 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 48 hours.

3.3.2 Preparation of the PI3V Positive Control Working Dilution

1. On the day of test initiation a vial of PI3V Positive Control is rapidly 

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

50 to 300 50% tissue culture infectious doses (TCID50) per 25 μL. Prior 

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

300 TCID50 for testing. This becomes the PI3V Working Dilution.

2. The PI3V 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 PI3V 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 Section 3.3.2(2.c) to the remaining tube, transferring 500 

μL from the previous dilution.
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3.3.3 Preparation of PSC and NSC

On day of test initiation, serial twofold dilutions of inactivated PSC and NSC are 

made from the original whole sera in a 96-well tissue culture plate labeled as the 

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.

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 PSC to wells A11 and B11, and 

150 μL of 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 the 

tips.

4. Using new pipettor tips, repeat Section 3.3.3(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. 

3.3.4 Preparation of 0.5% RBC suspension for HAd test

1. Upon receipt of the RBCs, transfer 20 mL of RBCs to a 50-mL conical 

tube.

2. Q.S. to 50 mL with Alsever’s Solution.

3. Mix by inverting several times.

4. Centrifuge for 15 ± 5 minutes at 400 x g (1500 rpm in the J6B 

centrifuge with a JS-4.0 rotor).

5. Remove supernatant and buffy coat by aspirating with a 10-mL

serological pipette.

6. Repeat Sections 3.3.4(2) through 3.3.4(4) for a total of 3 washes.
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7. Pipette 500 μL of packed RBCs to 100 mL of 1x PBS; mix by 

inverting for a 0.5% RBC suspension

8. Store at 4° ± 2°C; use within one week of collection of RBCs.

3.4 Preparation of the sample

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

postserums may be 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 in Sections 3.3.3(1) through 3.3.3(4).

4. Performance of the Test

4.1 On day of test initiation, dispense 150 μL of the PI3V Working Dilution to each 

well of the Control and Test Serum Transfer Plate using new pipette tips. Mix by gently 

tapping the sides each Transfer Plate. The addition of the PI3V Working Dilution to the 

wells results in an additional twofold serum dilution (final serum dilution).

4.2 Incubate the Transfer Plates at 36°± 2°C for 60 ± 10 minutes in a CO2 incubator.

4.3 Aseptically decant Growth Medium from the MDBK Test Plates into a suitable 

container.

4.4 Using an 8 channels of an 8-channel or 12-channel multipipettor, inoculate 

50 μL/well of the virus-serum mixture into 5 wells for each dilution of the Test Serum 

into the MDBK Test Plate (see 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 becomes the PI3V Test Plate.

4.5 Inoculate 5 wells/dilution with 50 μL/well of both the PSC and NSC.

4.6 Inoculate 5 wells/dilution with 25 μL/well of the PI3V Working Dilution and 

Back Titrations.

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

4.8 Incubate the PI3V Test Plates at 37°± 1°C in a CO2 incubator for 60 ± 10 minutes

for adsorption.
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4.9 After incubation, add 200 μL/well of Maintenance Medium to the PI3V Test 

Plates with a multichannel pipettor.

4.10 Incubate the PI3V Test Plates undisturbed at 37°± 1°C in a CO2 incubator for 4 

days ± 6 hours.

4.11 At the end of incubation, read the PI3V Test Plates at 100X magnification on an 

inverted light microscope and examine for CPE characterized by cell fusion.

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

CPE negative (CPE -) wells versus total number of wells examined for each 

dilution.

4.11.2 Results of the PI3V Working Dilution and Back Titration are recorded as 

the number of CPE positive (CPE +) wells versus total number of wells examined 

for each dilution.

4.12 If CPE is not detected by microscopic examination, the PI3V Test Plates may be 

read by HAd as follows:

4.12.1 Decant the Maintenance Medium from the PI3V Test Plates into a suitable 

autoclavable container and rinse cells once with 1X PBS at room temperature

using a plastic wash bottle or by immersion into a pan with 1X PBS at room 

temperature. Decant the 1X PBS immediately after filling.

4.12.2 Add 200 μL of the 0.5% RBC Suspension to each well.

4.12.3 Incubate the plates for 15 ± 5 minutes at room temperature.

4.12.4 Decant the 0.5% RBC suspension and wash the cell monolayer 3 times 

with 1X PBS as in Section 4.12.1.

4.12.5 Decant the final wash and examine the monolayers using an inverted light 

microscope at 100X magnification. Wells containing one or more RBC clusters 

adhering to the cell monolayer are considered to be positive for PI3V.

4.13 Calculate each endpoint of the Test Serum, PSC, and NSC using the SpearmanKärber method as commonly 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 PI3V.
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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 - (13/5=2.6)

Titer = (0.3 - 0.3/2 + [0.3 * 13/5]) = 0.93

antilog of 0.93 = 8.5

Titer of the Test Serum is 1:9

4.14 By the same Spearman-Kärber method, calculate the endpoint of the PI3V Back 

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

25 μL dose.

Example:

100 dilution of the PI3V Back Titration = 5 of 5 wells CPE positive

10-1dilution of the PI3V Back Titration = 5 of 5 wells CPE positive

10-2dilution of the PI3V Back Titration = 3 of 5 wells CPE positive

10-3dilution of the PI3V Back Titration = 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 + (13/5=2.6)

Titer = (0 - d/2 + [1 * 1.6]) = 2.1

antilog of 2.1 = 125.9

Titer of the PI3V Back Titration 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 without prejudice.

 
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5.1.1 The NSC must have an SN titer of < 1:2.

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 PI3V Back Titration titer must be between 50 to 300 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 (fourfold increase from the prevaccination titers).

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, NY, pg. 731.

8. Summary of Revisions

Version .05

 2.2.6, 2.2.7, 2.2.10: NCAH media numbers added.

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.

 References to NVSL have been changed to NCAH throughout 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: 

 Joseph Hermann has been added as a Contact for this document.

 1: Primary bovine embryonic kidney cell cultures have been replaced by MadinDarby bovine kidney-A cells.

 2.2.3: The description of antibiotics added to the Diluent Medium has been changed 

to reflect antibiotics used. Penicillin and streptomycin have been replaced by 

gentamicin sulfate, and amphotericin B has been eliminated from the formulation.
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 2.2.10: The 24-well cell culture plates have been replaced by 96-well plates.
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Appendix I

Dilution/Transfer Plate

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

A 

1:2

TS1 TS2 TS3 TS4 TS5 TS6 TS7 TS8 TS9 TS10 TS11 TS12

B 

1:4

↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓

C 

1:8

↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓

D

1:16

↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓

E 

1:32

↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓

F 

1:64

↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓

G 

1:128

↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓

H 

1:256

↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓

TS= Pre- or Post-Test Serum

Control/Transfer Plate

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

A 

1:2

PCS NC

S

TS TS TS TS TS TS TS TS TS TS 

B 

1:4

↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓

C 

1:8

↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓

D

1:16

↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓

E 

1:32

↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓

F 

1:64

↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓

G 

1:128

↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓

H 

1:256

↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓

PCS = Positive Control Serum

NCS = Negative Control Serum

TS = Test Serum
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Appendix II

PI3V 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 

1:4

↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓

C 

1:8

↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓

D

1:16

↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓

E 

1:32

↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓

F 

1:64

↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓

G 

1:128

↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓

H 

1:256

↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓

TS= Test Serum

CC= Cell Control
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PI3V Control Test Plate

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

A 

1:2

PCS PCS PCS PCS PCS CC NC

S

NC

S

NC

S

NC

S

NC

S

CC

B 

1:4

C 

1:8

D 

1:16

E 

1:32

WD

100

dil

WD

100

dil

WD

100

dil

WD

100

dil

WD

100

dil

F 

1:64

WD

10-1

dil

WD

10-1

dil

WD

10-1

dil

WD

10-1

dil

WD

10-1

dil

G 

1:128

WD

10-2

dil

WD

10-2

dil

WD

10-2

dil

WD

10-2

dil

WD

10-2

dil

H 

1:256

WD

10-3

dil

WD

10-3

dil

WD

10-3

dil

WD

10-3

dil

WD

10-3

dil

PCS = Positive Control Serum

NCS = Negative Control Serum

CC = Cell Control

WD = PI3V Back Titration