Hemagglutination assay
Encyclopedia
The hemagglutination assay (or haemagglutination assay; HA) is a method of quantification for virus
es or bacteria
by hemagglutination
. It is an easy, simple and rapid method which can be applied to large numbers of samples.
Some viral families and many bacteria have envelope or surface protein
s which are able to agglutinate (stick to) human
or animal red blood cell
s (RBC) and bind to N-acetylneuraminic acid
. As each of the agglutinating molecule attaches to multiple RBCs, a lattice-structure will form.
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.
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.
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 4C, otherwise viruses with neuraminidase activity will detach the virus from the RBCs. Then the lattice forming parts will be counted and the titer calculated.
Virus concentration in virions per milliliter
= 107 x HA titer
.
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.
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.
Virus
A virus is a small infectious agent that can replicate only inside the living cells of organisms. Viruses infect all types of organisms, from animals and plants to bacteria and archaea...
es or bacteria
Bacteria
Bacteria are a large domain of prokaryotic microorganisms. Typically a few micrometres in length, bacteria have a wide range of shapes, ranging from spheres to rods and spirals...
by hemagglutination
Hemagglutination
Hemagglutination, or haemagglutination, is a specific form of agglutination that involves red blood cells . It has two common uses in the laboratory: blood typing and the quantification of virus dilutions.-Blood Typing:...
. It is an easy, simple and rapid method which can be applied to large numbers of samples.
Some viral families and many bacteria have envelope or surface protein
Protein
Proteins are biochemical compounds consisting of one or more polypeptides typically folded into a globular or fibrous form, facilitating a biological function. A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of...
s which are able to agglutinate (stick to) human
Human
Humans are the only living species in the Homo genus...
or animal red blood cell
Red blood cell
Red blood cells are the most common type of blood cell and the vertebrate organism's principal means of delivering oxygen to the body tissues via the blood flow through the circulatory system...
s (RBC) and bind to N-acetylneuraminic acid
N-Acetylneuraminic acid
N-Acetylneuraminic acid is the predominant sialic acid found in mammalian cells.This negatively charged residue is found in complex glycans on mucins and glycoproteins found at the cell membrane...
. As each of the agglutinating molecule attaches to multiple RBCs, a lattice-structure will form.
The hemagglutination assay of a virus, in contrast to other forms of virus quantification
Virus Quantification
Virus quantification involves counting the number of viruses in a specific volume to determine the virus concentration. It is utilized in both research and development in commercial and academic laboratories as well as production situations where the quantity of virus at various steps is an...
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.
The detailed conditions depend on the type of virus or bacteria being assayed since certain pH
PH
In chemistry, pH is a measure of the acidity or basicity of an aqueous solution. Pure water is said to be neutral, with a pH close to 7.0 at . Solutions with a pH less than 7 are said to be acidic and solutions with a pH greater than 7 are basic or alkaline...
values and ionic strengths can impact the activity of the proteins of interest in a difficult to predict manner.
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 4C, otherwise viruses with neuraminidase activity will detach the virus from the RBCs. Then the lattice forming parts will be counted and the titer calculated.
Virus concentration in virions per milliliter
Litre
pic|200px|right|thumb|One litre is equivalent to this cubeEach side is 10 cm1 litre water = 1 kilogram water The litre is a metric system unit of volume equal to 1 cubic decimetre , to 1,000 cubic centimetres , and to 1/1,000 cubic metre...
= 107 x HA titer
Titer
A titer is a way of expressing concentration. Titer testing employs serial dilution to obtain approximate quantitative information from an analytical procedure that inherently only evaluates as positive or negative. The titer corresponds to the highest dilution factor that still yields a positive...
.
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.
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
Bacterial growth
250px|right|thumb|Growth is shown as L = log where numbers is the number of colony forming units per ml, versus T Bacterial growth is the division of one bacterium into two daughter cells in a process called binary fission. Providing no mutational event occurs the resulting daughter cells are...
before being observed.