Haemagglutination inhibition test (HA test)



 

All serological tests are based on the Ag-Ab reaction. It is also a serological test.

Principle

Ag or virus binds with Abs and causes the inhibition of HA.
We take higher titer of virus.

Objective of the test

  1. To check the level of Abs in flock.
  2. We can evaluate our vaccine result.
  3. We can check the typing of the HA virus.

Material

  • Normal saline or PBS (phosphate buffer saline) for dilution of serum.
  • Serum or antisera which contains Abs.
  • Ag from 4 HA.
  • Washed RBCs.

Preparation of serum

Serum is collected for testing the presence of Ag.
  1. Collect blood without anticoagulants.
  2. Keep blood tilted undisturbed at room temperature.
  3. Collect opaque fluid and centrifuge it.
  4. Safe supernatant and discard sediment.

Procedure of HI test

  1. Pour 25 ul PBS in all the wells of microtitration plates.
  2. Add 25 ul serum in these wells up to 11th wells by 2 fold serial dilution. In the 12th well, we have undiluted serum. 12th well has 25 ul PBS and 25 ul serum.
  3. Add 25 ul of 4 HA titer Ag up to 11th well.
  4. Incubate the plate for at least 30 minutes at room temperature.
  5. After the incubation period, add 25 ul washed RBCs and again incubate at least for 45 minutes.

Interpretation

Ag-Ab reaction          =>     button formation          conc. = 1/128
HA                             =>     clumping without Ag-Ab reaction.
  • If conc. is less than 1/128 then we increase our vaccine.
  • Button formation in 1/128 conc. well shows that our flock has enough amount of Ab to tackle the disease.

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