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SAM 124.05

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United States Department of Agriculture

Center for Veterinary Biologics

Testing Protocol

SAM 124

Supplemental Assay Method for Conducting the Hemagglutination Inhibition 

Assay for Equine Influenza Antibody

Date: October 23, 2014 Approved, Pending Standard Requirement

Number: SAM 124.05

Supersedes: SAM 124.04, September 9, 2011

Standard Requirement:

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

Sandra K. Conrad

Peg A. Patterson

Approvals: /s/Geetha B. Srinivas Date: 02Dec14

Geetha B. Srinivas, Section Leader

Virology

/s/Byron E. Rippke Date: 03Dec14

Byron E. Rippke, Director

Policy, Evaluation, and Licensing

Center for Veterinary Biologics

/s/Rebecca L.W. Hyde Date: 08Dec14

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.
Center for Veterinary Biologics SAM 124.05

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Supplemental Assay Method for Conducting the Hemagglutination Inhibition Assay 

for Equine Influenza Antibody

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Table of Contents

1. Introduction

2. Materials

2.1 Equipment/instrumentation

2.2 Reagents/supplies

3. Preparation for the Assay

3.1 Personnel qualifications/training

3.2 Preparation of reagents/control procedures

3.3 Preparation of the sample

4. Performance of the HI Assay

5. Interpretation of the HI Results

6. Report of the HI Results

7. References 

8. Summary of Revisions

Appendices
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Supplemental Assay Method for Conducting the Hemagglutination Inhibition Assay 

for Equine Influenza Antibody

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1. Introduction

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

hemagglutination inhibition (HI) antibody titer of sera from guinea pigs vaccinated with type A 

equine influenza viruses as part of a potency test for veterinary vaccines.

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 Micropipettors: 200-μL and 1000-μL single channel, 5- to 50-μL x 

12-channel, and tips

2.1.2 Centrifuge and rotor (Model J6-B centrifuge and Model JS-4.0 rotor, 

Beckman Instruments Inc.)

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

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 Round-bottom plate, 96-well

2.2.2 Polystyrene tube, 12 x 75-mm

2.2.3 Conical tube, 50-mL

2.2.4 Pipettes, 2-mL and 25-mL

2.2.5 Reagent reservoir

2.2.6 0.01 M Phosphate buffered saline (PBS) National Centers for Animal 

Health (NCAH) Media #30054
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Supplemental Assay Method for Conducting the Hemagglutination Inhibition Assay 

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1. 1.19 g sodium phosphate, dibasic, anhydrous (Na2HPO4)

2. 0.22 g sodium phosphate, monobasic, monohydrate (NaH2PO4•H2O)

3. 8.5 g sodium chloride (NaCl)

4. Q.S. to 1000 mL with distilled water (DW).

5. Adjust pH to 7.2-7.6 with 0.1 N sodium hydroxide (NaOH) or 1.0 N 

hydrochloric acid (HCl).

6. Sterilize by autoclaving at 15 psi, 121°± 2°C for 35 ± 5 minutes.

7. Store at 2°- 7°C.

2.2.7 10% Kaolin Suspension

1. 10 g kaolin in 100 mL PBS

2. Store at 2°- 7°C.

2.2.8 Alsever’s Solution

1. 8.0 g sodium citrate (C6H5Na3O7•2H2O)

2. 0.55 g citric acid (C6H8O7•H2O)

3. 4.2 g NaCl

4. 20.5 g glucose

5. Q.S. to 1000 mL with DW.

6. Sterilize with a 0.22-μm filter.

7. Store at 2°- 7°C.

2.2.9 Chicken red blood cells (RBC) from specific-pathogen-free chickens in an 

equal volume of Alsever’s Solution. Store at 2°- 7°C.
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Supplemental Assay Method for Conducting the Hemagglutination Inhibition Assay 

for Equine Influenza Antibody

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2.2.10 Test Virus

Each manufacturer provides each strain of type A equine influenza virus present 

in the Test Serial that has been correlated to protection in a host animal 

immunogenicity trial. Each Test Virus is identified in the Animal and Plant 

Health Inspection Service (APHIS) filed Outline of Production.

2.2.11 Equine influenza virus reference antiserum (Reference Serum) to serve as 

a positive control

3. Preparation for the Assay

3.1 Personnel qualifications/training

Personnel shall have training in hemagglutination (HA) and HI techniques and in 

standard laboratory procedures.

3.2 Preparation of reagents/control procedures

3.2.1 Upon receipt of the RBC, prepare Washed RBC as follows:

1. Transfer 20 mL of RBC into a 50-mL conical tube.

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

3. Mix by inverting several times.

4. Centrifuge for 10 minutes at 400 X g (1500 rpm in the J6-B centrifuge 

with a JS-4.0 rotor).

5. Remove supernatant and white blood cell layer by aspirating with a 

25-mL pipette.

6. Repeat Steps 2 through 5 for a total of 3 washes.

7. Store the packed RBC in Alsever’s Solution at 2°- 7°C; use within 1 

week of collection of RBC.

3.2.2 0.5% RBC Suspension for the HA or HI assays

Pipette 500 μL of packed Washed RBC into 100 mL of PBS. Store at 2°- 7°C; use 

within 1 week of collection of RBC.
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Supplemental Assay Method for Conducting the Hemagglutination Inhibition Assay 

for Equine Influenza Antibody

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3.2.3 5% RBC Suspension for removal of nonspecific agglutinins from 

guinea pig sera (GPS)

Pipette 100 μL of packed Washed RBC into 1.9 mL of PBS. Store at 2°- 7°C; use 

within 1 week of collection of RBC.

3.2.4 Test Virus Working Dilution

Each Test Virus should contain 4-8 HA units (HAU) per 25 μL. The Test Virus 

Working Dilution is determined by an HA assay and verified by back titration for 

each HI test performed.

On the day of test initiation, prepare a series of twofold dilutions of each Test 

Virus in duplicate from undiluted through 1:2048 in a 96-well, round-bottom plate 

(see Template, Appendix I). The same pipette tips may be used throughout the 

dilution scheme.

1. Pipette 50 μL/well of PBS into columns 2-12 with a 12-channel 

micropipettor.

2. Thaw frozen Test Virus at room temperature. Add 100 μL of undiluted 

Test Virus to wells A1 and B1. Additional Test Viruses are tested in 

duplicate in rows C through F.

3. Transfer 50 μL from column 1 to column 2 with a 12-channel 

micropipettor; mix (7 ± 2 fills by aspiration and expulsion of the 12-

channel micropipettor).

4. Continue Step 1.c for columns 3-12, transferring 

50 μL from the previous well to next well in the column until the dilution 

sequence is completed. Remove and discard 50 μL from the last column.

5. Add 50 μL of 0.5% RBC to each well containing Test Virus.

6. Place 50 μL of the 0.5% RBC Suspension in each of 3 wells as an 

RBC Control. Add 50 μL of PBS to each of the 3 RBC Control wells.

7. Mix by tapping the edge of the plate with fingers. Incubate the plate 

uncovered for 35 ± 10 minutes at room temperature. The optimum time to 

evaluate results is when distinct buttons have formed in the RBC Control 

wells. 
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Supplemental Assay Method for Conducting the Hemagglutination Inhibition Assay 

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8. Read the assay as follows: Record “+” for complete agglutination 

(hazy appearance to the RBC) and “-” for no agglutination (distinct 

button) and “ ±” for incomplete agglutination. To confirm specificity of 

agglutination, tilt the plate at a 45o angle for 20-30 seconds. If the settled 

RBCs “run” or form a tear-drop appearance, that particular well is 

considered to be negative for agglutination. 

9. The endpoint HA titer is considered to be the highest dilution of virus 

that completely agglutinates the RBCs. Wells with partial (incomplete) 

agglutination are not considered when determining endpoint values. The 

RBC diluent control wells should have distinct buttons. The endpoint HA 

titer is considered to be the dilution of Test Virus that contains 1 HA unit 

(HAU) per 50 μL. A virus concentration of 4-8 HAU per 25 μL is used for 

the HI assay. In order to determine a dilution factor of the Test Virus 

containing 4-8 HAU per 25 μL, the endpoint dilution is divided by 12. For 

example, if the endpoint titer of the Test Virus is 256, the dilution 

containing 4-8 HAU per 25 μL would be approximately 1:20 (256 divided 

by 12). The concentration of virus in this preparation (working dilution) 

should be confirmed by a virus back titration prior to performing the HI 

assay and is repeated when the HI portion of the assay is conducted.

3.3 Preparation of the sample

3.3.1 Guinea pig sera from test animals may be stored frozen indefinitely at 

-20°± 2°C. Treated serum may be stored at 2°- 7°C for up to 48 hours, or frozen 

indefinitely at -20°± 2°C. 

3.3.2 Treatment of GPS samples for the removal of nonspecific inhibitors

1. On the day of test initiation, pipette 200 μL of each GPS into 

individually labeled 12 x 75-mm polystyrene tubes, one tube for each 

serum to be tested.

2. Pipette 1.0 mL of 10% Kaolin Suspension (keep kaolin in suspension 

by periodic shaking) to each tube.

3. Vortex the kaolin/serum mixture tubes on high speed to resuspend the 

kaolin every 5 minutes for 20 ± 5 minutes.

4. Centrifuge at 800 X g (2000 rpm in the J6-B centrifuge with a JS-4.0 

rotor) for 20 ± 5 minutes.

5. Pipette 100 μL 5% RBC to each tube.
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Supplemental Assay Method for Conducting the Hemagglutination Inhibition Assay 

for Equine Influenza Antibody

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6. Pipette 700 μL PBS to each tube.

7. Gently shake the rack of tubes every 5 minutes for 20 ± 5 minutes to 

keep the RBCs in suspension. The kaolin pellet should not be disturbed by 

this treatment.

8. Centrifuge at 400 X g (1500 rpm in the J6-B centrifuge with a JS-4.0 

rotor) for 20 ± 5 minutes. The RBC pellet will pack down on top of the 

kaolin pellet.

9. Pour the supernatant from each treated GPS to a clean, labeled 12 x 

75-mm polystyrene tube. This is considered to be a 1:10 dilution of 

Treated GPS.

4. Performance of the HI Assay

4.1 Pipette 50 μL each of the Treated GPS into the first well of 2 adjacent columns on 

a 96-well, round-bottom plate for each Test Virus (see Template, Appendix II). This is 

the Sample Test Plate.

4.2 Pipette 25 μL of PBS into the remaining wells of each column with the 12-

channel micropipettor.

4.3 Twofold dilutions are made as follows:

4.3.1 Transfer 25 μL of serum from row 1 to row 2; mix (7 ± 2 fills by 

aspiration and expulsion of the 12-channel micropipettor).

4.3.2 Continue the 25-μL transfer until all dilutions have been made through 

row 8. Remove and discard 25 μL from the last row.

4.4 Add 25 μL of the Test Virus Working Dilution (4-8 HAU/25 μL) (Section 3.2.4) 

to each well.

4.5 Virus Back Titration is performed for each Test Virus (see Template, Appendix 

II).

4.5.1 Add 100 μL of the Test Virus Working Dilution in the first wells of 2 

adjacent columns of the Sample Test Plate (such as A11 and A12).

4.5.2 Add 50 μL of PBS in each of the 5 wells in the rows below the first wells 

of the 2 adjacent columns (B11 and B12 through F11 and F12).
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Supplemental Assay Method for Conducting the Hemagglutination Inhibition Assay 

for Equine Influenza Antibody

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4.5.3 Transfer 50 μL from duplicate wells of row 1 to row 2; mix (7 ± 2 fills by 

aspirating and expulsion of the 12-channel micropipettor).

4.5.4 Continue Step 5.3 for rows 3 through 6, transferring 50 μL from the 

previous wells to the next wells in the column. Remove and discard 50 μL from 

the last row used.

4.6 Place 25 μL of each GPS in an individual well as an autoagglutination control 

(GPS Autoagglutination Control). Add 25 μL of PBS to each of these GPS control wells.

4.7 Mix by tapping the edge of the plate with fingers. Incubate the plate uncovered 

for 30 ± 5 minutes at room temperature.

4.8 Add 50 μL of PBS to each of the 3 RBC Control wells and place 50 μL of the 

0.5% RBC Suspension in each of the 3 wells. 

4.9 Add 50 μL of 0.5% RBC to each well containing the Treated GPS/Test Virus 

mixture, the Virus Back Titration, or the GPS Autoagglutination Control.

4.10 Mix by tapping the edge of the plate with fingers. Incubate the plate uncovered 

for 35 ± 10 minutes at room temperature. The optimum time to evaluate results is when 

distinct buttons have formed in the RBC Control wells.

4.11 Read the agglutination results as follows:

“+” partial to complete agglutination (hazy appearance) indicates absence of 

antibody in the GPS.

“-” no agglutination (distinct button) indicates presence of antibody in the GPS.

4.12 The HI titer of the GPS is the reciprocal of the highest serum dilution with a 

distinct button of RBC in both test wells.

4.13 Group the test sera based on the vaccination serial. Determine the geometric mean 

titer (GMT) for each Test Virus from each group.

5. Interpretation of the HI Results

5.1 Validity requirements

5.1.1 The Virus Back Titration must show complete agglutination in the first 3 

dilutions, partial to full agglutination in the 4th dilution, and weak partial to no 

agglutination in the 5th and 6th dilution.
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Supplemental Assay Method for Conducting the Hemagglutination Inhibition Assay 

for Equine Influenza Antibody

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5.1.2 For a given fraction, the unvaccinated GPS must remain seronegative at 

the 1:10 dilution.

5.1.3 None of the GPS Autoagglutination Control wells may exhibit 

autoagglutination.

5.1.4 The titer of the Reference Serum must fall within ± 1 dilution of its 

recorded titer, as determined by a minimum of 10 previous titrations.

5.1.5 If these validity requirements are not met, the test is NO TEST and may 

be repeated.

5.2 If the GMT of the GPS of a Test Serial for a given fraction in an initial valid test 

is equal to or greater than the requirements stated in the APHIS filed Outline of 

Production, the Test Serial is SATISFACTORY.

5.3 If the GMT of the GPS of a Test Serial in an initial valid test, for a given fraction, 

is less than the GMT specified in the APHIS filed Outline of Production, the Test Serial 

may be retested in an equivalent number of additional animals as the initial test tested.

5.3.1 If the GMT of the GPS from all vaccinated animals of a Test Serial in the 

initial valid test and the valid retest, for a given fraction, is equal to or greater than 

the requirements stated in the APHIS filed Outline of Production, the Test Serial 

is SATISFACTORY.

5.3.2 If the GMT of the GPS from all vaccinated animals of a Test Serial in the 

initial valid test and the valid retest, for a given fraction, is less than the GMT 

specified in the APHIS filed Outline of Production, the Test Serial is 

UNSATISFACTORY.

6. Report of the HI Results

Record all test results on the test record.

7. References

7.1 Conrath TB. Handbook of Microtiter Procedures. Clinical and Research 

Applications Laboratory, Cooke Engineering Co., Alexandria, Virginia, 1972.

7.2 Snedecor GW and Cochran WG. Statistical Methods, 6th ed., Iowa State 

University Press, Ames, Iowa, Chpt 11, 1967.
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8. Summary of Revisions

Version .05

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

to keep the same next review date for the document.

Version .04

 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 .03

 The document number has been changed from VIRSAM0124 to SAM 124.

 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 were made that impact 

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

 3.2.4(8) Clarification of agglutination specificity confirmation has been added.

 3.2.4(9) Clarification of endpoint HA titers has been added.

 The refrigeration temperatures have been changed from 4°± 2°C to 2°- 7°C. This 

reflects the parameters established and monitored by the Rees system.
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Supplemental Assay Method for Conducting the Hemagglutination Inhibition Assay 

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Appendix I

Standardization of Test Viruses Plate

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

A A 1

B A 1

C A 2

D A 2

E A 3

F A 3

G UN 1:2 1:4 1:8 1:16 1:32 1:64 128 256 512 1024 2048

H RBC RBC RBC

A 1= Type A Influenza Isolate No. 1, etc. RBC= RBC Control

Appendix II

Sample Test Plate

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

A

1:10

GPS

1

GPS

1

GPS

2

GPS

2

GPS

3

GPS

3

GPS

4

GPS

4

GPS

5

GPS

5

BT BT

B

1:20

1:2 1:2

C

1:40

1:4 1:4

D

1:80

1:8 1:8

E

1:160

1:16 1:16

F

1:320

1:32 1:32

G

1:640

RBC RBC

H

1:1280

RBC RBC

GPS= Treated Guinea Pig Serum

BT= Back Titration of the Test Virus Working Dilution

RBC= RBC Control