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Laboratory Exercise 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)

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Laboratory Exercise Detection and Identification of Hemagglutinating Isolates

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  1. 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 TestsSupplies 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

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

  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

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

  15. 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

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

  17. HI – InterpretationIsolates • 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

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

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

  20. 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

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