Detection and Identification of Hemagglutinating Isolates 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) Single channel (calibrated) – 25-200ul Multichannel (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 2 128 256 64 32 16 8 4 1 +C 3 5 6 7 9 10 CC

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

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

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 1 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

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

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

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

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