Question

The undigested antibodies and pepsin digested antibodies were tested for haemagglutination. In the haemagglutination assay, the titre for undigested antibodies is 8 and pepsin digested antibodies is 4. why pepsin digested abs has lower titre compared to undigested abs. State 2 reason.

Answer 1

What is a haemagglutination test?

This assay uses the ability of viruses t bind with the RBC with their specific proteins. Thus by analyzing the reaction made on RBC, we can quantify the amount of virus present in the sample. If a person is affected with the virus, the virus form lattices and the blood seems agglutinated. By analyzing the presence of agglutination up tp which dilution gives the amount of virus present. If the virus is not present in the sample, then no lattice is formed. The unit used to evaluate viral quantity is HAU. The reciprocal of endpoint value is called haemagglutination titre (HA titre).

· If the virus is too many in the sample, then the antibody cannot interact with all viruses. Thus the virus interact with RBC and form agglutination in higher dilutions.

· If virus is too low, then antibody will interact with viral antigen and no agglutination is formed with RBC.

The endpoint dilution upto which virus can form agglutination with RBC depends on the viral quantity and also to the efficiency of the antibody present.

Antibody digestion and its advantages?

Digested antibodies have many advantages over undigested antibodies. They are,

· Fc interactions are reduces as it is cleaved which may make non specific interactions which alter the result.

· Fab can efficiently bind to the antigen.

· Has lower immunogenicity than undigested antibodies.

Pepsin is a non-specific endonuclease that cleaves antibodies. It forms 2 Fab fragments. Its advantage is that its binding site is always exposed. So it can bind more effectively.

Thus , the undigested antibody is less efficient that it interact with less amount of virus and thus virus can form agglutination with RBC in more dilutions. But, pepsin digested antibody is more efficient that it can bind with more virus and thus very less amount of virus can interact with RBC and form agglutination.

That’s why, undigested antibody having sample showed agglutination upto 1/8 dilution and gave 8HA titre. But digested antibody showed endpoint in the ¼ dilution. Thus had a HA titre of 4.

This is just about the binding efficiency of antibody and the viral quantity remains more or less the same.