1. Detection and Identification of Hemagglutinating Isolates
Laboratory Exercise
Detection and Identification of Hemagglutinating Isolates

2. Hemagglutination (HA) Test

3. Hemagglutination (HA) Test+
RBC Suspension
HA Virus
Settling Pattern

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

5. 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

6. Use sterile aerosol-resistant tips (ART) when manipulating AAF from original isolate tube to avoid cross contamination of samples
USDA-APHIS

7. USDA-APHIS

8. USDA-APHIS

9. USDA-APHIS

10. Hemagglutination (HA) Test2
128
256 64 32 16 8 4 1 +C 3 5 6 7 9 10 CC

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

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

13. 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

15. Hemagglutination-Inhibition (HI) Test+ +
RBC Suspension
HA Virus
Antibody
Settling Pattern

16. 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

17. Hemagglutination-Inhibition (HI) Test1 10 2 3 4 5 6 7 8 9
Back Titration +C CC 2
128
256 64 32 16 8 4 1 3 5 6 7 9 +C 10 CC

18. HI – Interpretation Isolates
Avian Paramyxovirus Type-1 (APMV-1)
Endpoint within 2 wells (4-fold dilution) of APMV-1 positive control = APMV-1
Avian influenza
Any inhibition = positive for the subtype used
USDA-APHIS

19. Hemagglutination-Inhibition (HI) Test
Interpretation (APMV-1)
APMV-1
Neg APMV-1 (may be AIV) 2
128
256 64 32 16 8 4 1 3 5 6 7 9 +C 10 CC

20. AIV HI Screen Setup H1 H2 H3 H4 1 2 3 4 5 6 7 +C

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