The hemagglutination assay (or haemagglutination assay; HA) is a method of quantification for viruses or bacteria by hemagglutination. It is an easy, simple and rapid method which can be applied to large numbers of samples. The hemagglutination assay and its extension, the hemagglutination inhibition assay, were invented in 1941–42 by American virologist George Hirst.
The hemagglutination assay of a virus, in contrast to other forms of virus quantification such as a plaque assay or 50% Tissue Culture Infective Dose, does not give any measure of viral infectivity, because no virus replication is required in this assay. The same may not be true when using HA for bacteria.
Some viral families and many bacteria have envelope or surface proteins which are able to agglutinate (stick to) human or animal red blood cells (RBC) and bind to N-acetylneuraminic acid. As each of the agglutinating molecule attaches to multiple RBCs, a lattice-structure will form.
The detailed conditions depend on the type of virus or bacteria being assayed since certain pH values and ionic strengths can impact the activity of the proteins of interest in a difficult to predict manner. Typically avian RBCs from chickens or turkeys are used in this assay as they settle fast and form a compact button in a V-well microtiter plate.
Normally, a virus dilution (e.g. 2-fold from 1:4 to 1:4096) will be applied to an RBC dilution (e.g. 0.1% to 0.7% in steps of 0.2%) for approx. 30 min, often at 4 °C, otherwise viruses with neuraminidase activity will detach the virus from the RBCs. Then the lattice forming parts will be counted and the titer calculated.
The titer of a hemagglutination assay is determined by the last viable "lattice" structure found. This is because it is at the point where, if diluted anymore, the amount of virus particles will be less than that of the RBCs and thus not be able to agglutinate them together. The HA titer is the reciprocal of the dilution in this well.
An HA unit (HAU) is the amount of virus needed to agglutinate an equal volume of standardized RBCs.
For bacteria, depending on species, a bacterial dilution will be applied to an equal part RBC dilution and then incubated for 30 min to an hour at an optimal growth temperature before being observed.
Hemagglutination inhibition assay
The hemagglutination inhibition assay is a common variation of the HA assay used to measure flu-specific antibody levels in blood serum. In this variation, serum antibodies to the hemmagglutinin (HA) surface glycoprotein will interfere with the virus attachment to red blood cells. Therefore hemagglutination is inhibited when antibodies are present at a sufficient concentration. In order to accurately perform this assay, nonspecific agglutinins in serum samples must be removed to avoid false-negative results.
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