In the name of God. Summer School Influenza Unit, Pasteur Institute of Iran summer 2012

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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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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IntroductionSum

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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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HemagglutinationSum

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Hemagglutination Sum

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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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Antibodies against the viral protein responsible for haemagglutination can prevent haemagglutination.

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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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Hemagglutination InhibitionSum

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Hemagglutination InhibitionSum

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Procedure

Inactivation of non-specific inhibitorsHeatingRDE (Receptor Destroying Enzyme)Potassium periodateAlabama methodTrypsinCaolinS

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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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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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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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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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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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ProcedureSum

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Documents

In the name of God. Summer School Influenza Unit, Pasteur Institute of Iran summer 2012