Laboratory Exercise

Detection and Identification of

Hemagglutinating Isolates
Hemagglutination (HA) Test
 Hemagglutination (HA) Test

RBC Suspension HA Virus Settling Pattern

 HA/HI Tests
Supplies Needed
• U- or V-bottom microtiter plates (non-sterile)
• Pipettors with tips (sterile ART, nonsterile)
9Single channel (calibrated) – 25-200ul
9Multichannel (calibrated) – 25-200ul
• Phosphate buffered saline (0.01M, pH 7.2)
• Washed chicken erythrocytes (0.5%)

USDA-APHIS
 HA Procedure

• Label plates (8 or 12 wells/row)

• Add 50 ul PBS to all wells
• Add 50 ul AAF to 1st well
• Mix & dilute (50 ul) – 1st through last well
• Add 50 ul 0.5% washed rbcs to all wells
• Mix by shaking plates
• Read at 20 – 30 minutes (when rbcs buttoned)
USDA-APHIS
Use sterile aerosol-resistant tips
(ART) when manipulating AAF
from original isolate tube to
avoid cross contamination of
samples USDA-APHIS
USDA-APHIS
USDA-APHIS
USDA-APHIS
Hemagglutination (HA) Test
2 4 8 16 32 64 128 256
1
2
3
4
5
6
7
8
9
10
+C
CC
 Hemagglutinating Units (HAU)

• 1 HAU = highest dilution giving complete HA

9Number of HAUs/50 ul = reciprocal of highest dilution
9e.g. an endpoint of 1:64 = 64 HAU/50 ul
• HI test for APMV – use 8 HAU/25 ul
9To calculate the dilution to achieve 8 HAU/0.025ml,
divide the HA endpoint by 16
9e.g. HA of 1:64, divided by 16 = 4 (dilute the isolates in
PBS 1:4 to obtain 8 HAU/25 ul)

USDA-APHIS
 Hemagglutination (HA) Test
Endpoint Dilution (8 HAU)
2 4 8 16 32 64 128 256
1 1:128 1:8
2 1:32 1:2
3 1:64 1:4
4 Neg
5 Neg
6 1:16 undiluted
7 Neg
8 1:32 1:2
9 1:128 1:8
10 Neg
+C
CC
 HA Procedure

• Label plates (8 or 12 wells/row)

• Add 50 ul PBS to all wells
• Add 50 ul AAF to 1st well
• Mix & dilute (50 ul) – 1st through last well
• Add 50 ul 0.5% washed rbcs to all wells
• Mix by shaking plates
• Read at 20 – 30 minutes (when rbcs buttoned)
USDA-APHIS
 Hemagglutination-Inhibition (HI) Test

+ +

RBC Suspension HA Virus Antibody Settling Pattern

 HI Procedure

• Add 25 ul diluted (8 HAU) AAF to all wells (rows

of 6 or 8 wells)
• Add 25 ul standardized antiserum (determined to
give endpoint of 1:32 with homologous antigen) to
1st well
• Dilute (25 ul) – 1st well through end of row
• Incubate 30 minutes at room temperature
• Add 50 ul 0.5% washed rbcs
• Read at 20 – 30 minutes
USDA-APHIS
 Hemagglutination-Inhibition (HI) Test
2 4 8 16 32 64 128 256 Back Titration
1 1
2 2
3 3
4 4
5 5
6 6
7 7
8 8
9 9
10 10
+C +C
CC CC
 HI – Interpretation
Isolates
• Avian Paramyxovirus Type-1 (APMV-1)
9Endpoint within 2 wells (4-fold dilution) of
APMV-1 positive control = APMV-1
• Avian influenza
9Any inhibition = positive for the subtype used

USDA-APHIS
Hemagglutination-Inhibition (HI) Test
2 4 8 16 32 64 128 256 Interpretation (APMV-1)
1 APMV-1
2 APMV-1
3 APMV-1
4 APMV-1
5 APMV-1
6 Neg APMV-1 (may be AIV)
7 Neg APMV-1 (may be AIV)
8 APMV-1
9 Neg APMV-1 (may be AIV)
10 Neg APMV-1 (may be AIV)
+C
CC
 AIV HI Screen Setup
H1 H2 H3 H4
1

6
7

+C
 Hemagglutinating Units (HAU)
AIV
• HI test for AIV – use 4 HAU
9To calculate the dilution to achieve 8
HAU/0.025ml, divide the HA endpoint by 8
9e.g. HA of 1:64, divided by 8 = 8 (dilute the
isolates in PBS 1:8 to obtain 4 HAU/25 ul)

USDA-APHIS