Methods

METHODS

Micro Haemagglutination Inhibition Test

  • Haemagglutination inhibition test can done in to ways.
    • Alpha method: Constant serum and varying virus concentration.
    • Beta method : Constant virus and varying serum concentration.
  • Among these two methods,Beta method is commonly used and is more accurate where as alpha method requires large amount of serum sample.

Procedure

  • Dispense 25 µl of PBS / normal saline into each well of the plates.
  • Shake each serum sample and dispense 25µl into the first well and the last  well (serum control) of a row of a micro well plate.
  • Use a micro pipette to make two-fold serial dilutions along the row until the second last well from the end. The last well is the serum control. Do not dilute this well.
  • Add 25 µl of the 4HA dilution of antigen to each well excluding the control wells
  • Gently tap the sides of the micro well plates to mix the reagents. Cover plates with a lid.
  • Allow for 30 minutes at room temperature.
  • Add 25 µl of 1 percent washed red blood cells to each well including the control wells in the last column.
  • Gently tap the sides of the micro well plates to mix the reagents. Cover the plates with a lid.
  • Allow standing at room temperature for 45 minutes.
  • RBC Control: Add 25 ul of saline to well to which add 25 ul of 1% chicken RBC
  • Check Virus Titration
    •  Add 25 ul of saline into four wells in a row.
    •  Add 25 ul of the 4HA virus suspension to the first well and make    serial two fold dilution.
    • Discard 25 ul of the suspension from the fourth well.
    • Add 25 ul of 1% chicken RBC to all the four wells.
    • Leave for 20 minutes.
    • The first two wells will show haemagglutination , if the viral suspension prepared is having 4HA units of virus in every 25 ul . 
    • Read the settling patterns for each serum sample.
    • Read the control serum well first then read the patterns in the other wells.
Last modified: Sunday, 25 September 2011, 10:13 AM