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
- To check the level of Abs in flock.
- We can evaluate our vaccine result.
- 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.- Collect blood without anticoagulants.
- Keep blood tilted undisturbed at room temperature.
- Collect opaque fluid and centrifuge it.
- Safe supernatant and discard sediment.
Procedure of HI test
- Pour 25 ul PBS in all the wells of microtitration plates.
- 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.
- Add 25 ul of 4 HA titer Ag up to 11th well.
- Incubate the plate for at least 30 minutes at room temperature.
- After the incubation period, add 25 ul washed RBCs and again incubate at least for 45 minutes.
Interpretation
Ag-Ab reaction => button formation conc. = 1/128HA => 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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