In the name of God
Summer School
Influenza Unit,Pasteur Institute of Iran summer 2012
Hemagglutination Inhibition assay
Dr V. Mazaheri
Introduction
A wide variety of different viruses possess the ability to agglutinate the erythrocytes of mammalian or avian species.
Some viral families have envelope or surface proteins which are able to agglutinate human or animal RBC and bind to N-acetylneraminic acid.
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Influenza Unit, Pasteur Institute of Iran summer 2012
Introduction
The surface proteins are the only antigens capable of inducing neutralizing antibody and therefore a protective immune response.
Antibodies against the HA protein are the main determinant for protection in the host against disease.
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Influenza Unit, Pasteur Institute of Iran summer 2012
IntroductionInfluenza hemagglutinin (HA) is an antigenic
glycoprotein. It is responsible for binding the virus to the cell that is being infected.
HA proteins bind to cells with sialic acid on the membranes, such as cells in the upper respiratory tract or erythrocytes.
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Influenza Unit, Pasteur Institute of Iran summer 2012
IntroductionSum
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Influenza Unit, Pasteur Institute of Iran summer 2012
HemagglutinationHemagglutination assay is a method for
titering influenza viruses based on their ability to attach to molecules present on the surface of red blood cells.
As each of the agglutinating molecule attaches to multiple RBCs, a lattice-structure will form.
A method of quantification for viruses by hemagglutination.
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Influenza Unit, Pasteur Institute of Iran summer 2012
HemagglutinationSum
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Influenza Unit, Pasteur Institute of Iran summer 2012
Hemagglutination Sum
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Influenza Unit, Pasteur Institute of Iran summer 2012
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HemagglutinationSum
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Influenza Unit, Pasteur Institute of Iran summer 2012
Hemagglutination Inhibition
Antibody titers to the HA protein are commonly measured by an indirect antibody test, the hemagglutinin inhibition (HI) test.
Hemagglutination Inhibition assay is a common variation of the HA assay used to measure antibody levels in blood serum.
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Influenza Unit, Pasteur Institute of Iran summer 2012
Hemagglutination Inhibition
Antibodies against the viral protein responsible for haemagglutination can prevent haemagglutination.
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Influenza Unit, Pasteur Institute of Iran summer 2012
Hemagglutination Inhibition
Simple to perform, economical, requires inexpensive equipment & reagents.
Reliable, time saving test.Is not sensitive as EIAs or RIAs.The actual reading of results is subjective .The reagents should be fresh.Serum treatment S
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Influenza Unit, Pasteur Institute of Iran summer 2012
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Influenza Unit, Pasteur Institute of Iran summer 2012
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Hemagglutination InhibitionSum
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Influenza Unit, Pasteur Institute of Iran summer 2012
Procedure
Inactivation of non-specific inhibitorsHeatingRDE (Receptor Destroying Enzyme)Potassium periodateAlabama methodTrypsinCaolinS
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Influenza Unit, Pasteur Institute of Iran summer 2012
Procedure
Inactivation of non-specific hemagglutininsRBC Preparation of standardized Ag An HA unit is defined as the amount of virus
needed to agglutinate an equal volume of a standardized RBC suspension.
The standard for the HAI test is 4 HA units of antigen added to 2-fold dilutions of antisera. S
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Influenza Unit, Pasteur Institute of Iran summer 2012
Procedure
Since we are adding 25 ml of antigen in the test, we need a virus dilution that contains
4 HA units/25 ml or 8 HA units/50 ml Calculate the antigen dilution by dividing the
HA titer, which is based on 25ml, and dividing by 4.
This titer will hemagglutinate the first four wells of the back titration plate. S
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Influenza Unit, Pasteur Institute of Iran summer 2012
Procedure
For example, an HA titer of 128 128 /4 =32 Mix 1 part of virus with 31 parts PBS to obtain
the desired volume of standardized antigen (Ex: add 0.1 ml antigen to 3.1 ml of PBS).
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Influenza Unit, Pasteur Institute of Iran summer 2012
Procedure
If an antigen does not titer 8, it must be adjusted accordingly by adding more antigen to increase units or by diluting to decrease units.
I) If complete hemagglutination is present in the fifth well, the virus now has a titer of 16 and the test antigen should be diluted two-fold. S
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Influenza Unit, Pasteur Institute of Iran summer 2012
Procedure
II) If hemagglutination is only present to the third dilution, the antigen has a titer of 4 and an equal volume of virus must be added to the test antigen as was used when the antigen was initially diluted. This will double the concentration of virus in the test antigen to give a titer of 8.
Continue adjusting the concentration of antigen until 4 HA units/ 25 ml is obtained.
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Influenza Unit, Pasteur Institute of Iran summer 2012
ProcedureSum
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Influenza Unit, Pasteur Institute of Iran summer 2012
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