Haemagglutination test (HA)


Objective

   HA test is used to determine the titer of viruses. It is also used to measure the 4 HA of antigen. It is also used to detect either the HA or HA virus.

Viral HA

NDV has protein neuraminidase. It helps in the clumping of RBCs.

Haemagglutinin

Any virus protein or enzyme that helps in clumping of RBCs.

Principle

Viruses are attached to the receptor of RBCs and results in agglutination or clumping.

Material

  1. Micro-titration plates (Round bottom or flat bottom, we use round bottom micro-titration plate for this test. Flat bottom micro-titration plates are used for biofilm detection)
  2. Allantoic fluid
  3. Micropipette
  4. 0.5% washed RBCs
  5. Test tube.

Procedure

  1. Collect 1 ml of blood from chicken and mix it with anticoagulant (EDTA).
  2. Add 2 ml of distilled water in the blood.
  3. Centrifuge test tube.
  4. RBCs settle at the bottom of the test tube, buffy coat (WBCs + platelets) at the middle, and the upper layer contains plasma.
  5. Collect RBCs and discard the supernatant.
  6. Repeat the above procedure with the same collected RBCs for 2-3 times.
  7. Now make 0.5 % RBCs solution by adding 0.5 mg RBCs and 99.5 ml distilled water.

Dilution of virus

  • Add 0.5 ul normal saline in all wells in 1st row of the microtitration plate.
  • Add 0.5 ul allantoic fluid in 1st well and make a serial dilution of 2 fold.

Serial dilution procedure

  • Mix normal saline and virus in well no. 1.
  • Transfer 0.5 ul to well no. 2 from well no. 1.
  • From well no. 2, transfer 0.5 ul to well no. 3 and repeat the same procedure upto 12th well.

Concentration

  • 1st well = 1/2, 2nd = 1/4, 3rd = 1/8, 4th = 1/16, 5th = 1/32, 6th = 1/64, 7th = 1/128 and so on.

After making 2 fold serial dilution

  • Add 0.5 % 0.5 ul RBCs in all the wells (1-12th).
  • Incubate plate at room temperature at least for 1 hour.
  • After every 15 minutes, we observe the result.
  • We observe clumping.
  • Where virus quantity is enough, clumping will occur and where virus quantity is not enough only button formation will occur.

Examples of agglutinating viruses

  • Paramyxovirus
  • Orthovirus
  • Adenovirus
  • Tobra virus

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