1. In the name of God

2. Summer School Influenza Unit, Pasteur Institute of Iran summer 2012

3. Hemagglutination Inhibition assay Dr V. Mazaheri

4. 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. Summer School Influenza Unit, Pasteur Institute of Iran summer 2012

5. 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. Summer School Influenza Unit, Pasteur Institute of Iran summer 2012

6. Introduction  Influenza 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. Summer School Influenza Unit, Pasteur Institute of Iran summer 2012

7. Introduction Summer School Influenza Unit, Pasteur Institute of Iran summer 2012

8. Hemagglutination  Hemagglutination 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. Summer School Influenza Unit, Pasteur Institute of Iran summer 2012

9. Hemagglutination Summer School Influenza Unit, Pasteur Institute of Iran summer 2012

10. Hemagglutination Summer School Influenza Unit, Pasteur Institute of Iran summer 2012 +

11. Hemagglutination Summer School Influenza Unit, Pasteur Institute of Iran summer 2012

12. 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. Summer School Influenza Unit, Pasteur Institute of Iran summer 2012

13. Hemagglutination Inhibition  Antibodies against the viral protein responsible for haemagglutination can prevent haemagglutination. Summer School Influenza Unit, Pasteur Institute of Iran summer 2012

14. 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 Summer School Influenza Unit, Pasteur Institute of Iran summer 2012

15. Hemagglutination Inhibition Summer School Influenza Unit, Pasteur Institute of Iran summer 2012 ++

16. Hemagglutination Inhibition Summer School Influenza Unit, Pasteur Institute of Iran summer 2012

17. Procedure Inactivation of non-specific inhibitors  Heating  RDE (Receptor Destroying Enzyme)  Potassium periodate  Alabama method  Trypsin  Caolin Summer School Influenza Unit, Pasteur Institute of Iran summer 2012

18. Procedure Inactivation of non-specific hemagglutinins  RBC 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. Summer School Influenza Unit, Pasteur Institute of Iran summer 2012

19. 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. Summer School Influenza Unit, Pasteur Institute of Iran summer 2012

20. 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). Summer School Influenza Unit, Pasteur Institute of Iran summer 2012

21. 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. Summer School Influenza Unit, Pasteur Institute of Iran summer 2012

22. 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. Summer School Influenza Unit, Pasteur Institute of Iran summer 2012

23. Procedure Summer School Influenza Unit, Pasteur Institute of Iran summer 2012

24. Thanks