The standard method used for the detection of influenza antibodies in animals is the haemagglutination inhibition (HI) test. Antibodies can also be detected by neutralisation test or ELISA.
HI is a serological test for the detection of antibodies which requires paired serum samples. It works on the principle that the haemagglutinin of influenza viruses, like that of other viruses, has a haemagglutinating effect on erythrocytes. This means that blood will clot on addition of these viruses, because the erythrocytes bind to the haemagglutinin on the surface of the virus.
Specific antibodies produced in response to infection or vaccination with the virus can inhibit this agglutination by binding to the haemagglutinins.
If the material for testing contains specific antibodies to the haemagglutinin, addition of the influenza virus produces an antigen/antibody reaction. This is made visible by the subsequent addition of erythrocytes. These are no longer agglutinated because the reaction is inhibited. The figure below illustrates the principle of the haemagglutination test and haemagglutination inhibition test.
Figure: Haemagglutination test and haemagglutination inhibition test.
The serum from pig "A" contains no antibodies to influenza, which is why haemagglutination occurs. The serum from pig "B" contains influenza antibodies, so agglutination is inhibited.
Serum titration can be used to determine the dilutions at which haemagglutination continues to be inhibited. The reciprocal value of the highest dilution at which inhibition of agglutination can still be observed gives the antibody titer (see figure right).
HI is a highly subtype-specific test. However, due to cross-reactions which may or may not occur within genetic subclusters of a subtype, it cannot identify all subclusters. Advantages of this method are its speed and convenience, and the minimal laboratory requirements.
Figure: Results of a haemagglutination inhibition test. The antibody titer is 1:80.
The neutralization test (NT) detects virus-specific neutralizing antibodies in the serum sample. A two-fold dilution of the serum sample and a known quantity of swine influenza virus are pre-incubated and then added to Madin-Derby canine kidney (MDCK) cells to determine the highest dilution, which can neutralize the virus infection, and the presence of a cytopathic effect (see figure below). An advantage of this method compared with HI is that it demonstrates the neutralizing activity of the serum antibodies. Drawbacks are the high laboratory requirements and the longer time taken to evaluate the results.
Figure: Neutralization test
Several ELISA test kits for the detection of a broad spectrum of anti-influenza A antibodies or the detection of antibodies to subtypes H1N1 and H3N2 are commercially available.