1 / 21

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)

dorian-yang
Download Presentation

Laboratory Exercise Detection and Identification of Hemagglutinating Isolates

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript


  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

More Related