Haemagglutination Tests Virtual Lab Simulation

General Aim of Haemagglutination Tests

Hemagglutination is used for the diagnosis of some enveloped viruses such as influenza viruses. 

Haemagglutination Tests Method

The Technique of Virtual Haemagglutination Test Haemagglutination test is used to quantify the amount of animal virus in a suspension. This is done by carrying out two-fold serial dilutions of the viral suspension in a microtitre plate or test tubes and then test to determine an end point. This result can then be used to determine the amount of haemagglutinin in the suspension and is expressed as a HA titre. One HA unit in the haemagglutinin titration is the minimum amount of virus that will cause agglutination of the red blood cells. 1. Prepare 10 sterile test tubes. 2. Add 0.5 ml of normal saline to each of the test tubes. 3. Add 0.5 ml of undiluted virus to the first tube ONLY and mix gently. 4. Transfer 0.5 ml to the next tube and mix gently. 5. Repeat this procedure for the whole test tubes, discarding 0.5 ml from the last tube (8th tube). 6. This will lead to bifold dilution of the virus starting from 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, 1:128 & 1:256. So the dilution in the 1st tube is 1:2 , in the 2nd tube is 1:4, in the 3rd tube is 1:8, in the 4th tube is 1:16, and so on. 7. Using a clean pipette tip, add 0.5 ml of 1% fresh RBCs suspension to each tube. 8. Two negative control test tubes (9th and 10th tubes) are prepared: one consisting of a mixture of normal saline and RBCs and the other of a mixture of non-infected allantoic fluid with the RBCs. 9. Allow the tubes to stand in ice for 45 minutes. 10. Read and record hemagglutination test results in each well. 11. Identify the end point. This will be the last well to show complete haemagglutination and contains one haemagglutinating unit.

Haemagglutination Tests Theory

Hemagglutination is used for the diagnosis of some enveloped viruses such as influenza viruses. This method relies on the specific feature of some enveloped viruses that can adsorb to red blood cells (RBCs). Specifically, hemagglutinin5 (HA), an envelope glycoprotein of some enveloped viruses, imparts this property. In the absence of virus particles, RBCs precipitate by gravity to the bottom of the well, giving rise to a distinct red-colored dot in a conical shaped well. In the presence of virus particles, RBCs clump together as a result of interaction between HA proteins of virus particles and RBC, leading to a lattice formation. In this case, as RBCs are dispersed as a clump, a red dot is not formed. 

In a given sample, a red dot will appear beyond a certain dilution fold. To carry out a hemagglutination assay, a twofold serial dilution of virus-containing samples is dispensed into individual wells of a 96-well microtiter plate. Then, aliquots of RBC are added to each well. The highest dilution at which clumping is observed is regarded as the HA titer of the sample. The virus titer in a sample can be estimated by multiplying the dilution fold. 
 

Haemagglutination Tests Principle

Hemagglutination is a classical method for viral diagnosis and it is still used for diagnosis of influenza virus today. One outstanding advantage of this method is that it does not require any equipment. Moreover, it is a robust and rapid diagnostic tool, but the sensitivity is somewhat limited.

Haemagglutination tests apply the process of hemagglutination, in which sialic acid receptors on the surface of red blood cells (RBCs) bind to the hemagglutinin glycoprotein found on the surface of influenza virus (and several other viruses) and create a network, or lattice structure, of interconnected RBC's and virus particles. The agglutinated lattice maintains the RBC's in a suspended distribution, typically viewed as a diffuse reddish solution. The formation of the lattice depends on the concentrations of the virus and RBC's, and when the relative virus concentration is too low, the RBC's are not constrained by the lattice and settle to the bottom of the well.