Infection & Immunity

Practical
MEASURING THE TITRE OF A VIRUS ANTIGEN USING A
HAEMAGGLUTINATION ASSAY

Mohammed Haseeb Chohan

Abstract:
Haemagglutination is a specific technique used when antibodies bind to red blood cells,
representing as an antigen. Its purpose is to tittering influenza viruses based upon their
ability to attach to molecules on the surface of red blood cells, specifically for blood
grouping or viral identification. PBS is serially diluted into a well of 96 well in which is
added a suspension of lectin (Lec1 and Lec2). The samples are mixed and set aside for
25 minutes. After this time, an estimation of the amount of virus present can be made.
This approach allows us to titre the sample being assessed using a swift and simple
technique.

Introduction:
Agglutination is when molecules in suspension such as antibodies are clump together in
cells or particles (Bunchanan, 1919). Virus have the ability to bind to RBCs and causes
agglutination. For example, Influenza viruses have a viral spike called hemagglutinin,
which has the capability to agglutinate red blood cells (Mögling et al., 2017). RBCs allows
the detection of certain influenza virus species by haemagglutination assays (Ryu, 2017).
Haemagglutination assay was developed by George Hirst (1941-1942) to quantify the
concentration of viruses in a sample and for epidemiological studies for examining
protection of susceptible populations (Hirst, 1942). In the haemagglutination assay, the
sialic acids on the surface of red blood cells (RBC) bind to the glycoprotein present on
the influenza virus. This create a lattice structure consisting of interconnected RBC and
virus particles (Centers for Disease Control and Prevention, 2017). When the virus
concentration is low, the RBC’s are not restricted by the lattice and settle at the bottom
of the well. If the serum of a person is infected with virus mixed with RBC’s, no
agglutination will occur (MacLachlan and Dubovi, 2016). This is because the antibodies
present in the serum of the infected person with the virus can be neutralised. Live virus
samples are replaced by Lectin. Lectin is a carbohydrate-binding protein which bind to
tissue specific glycans on the host cell-surface glycoproteins (Varki et al., 2009). The
basis of this assay is the ability of viral haemaglutinin to bind with the sialic acid present
on the receptors of surface of the red blood cells causing haemagglutination (Centers
for Disease Control and Prevention, 2017).

The purpose of the experiment is the estimate the number of virus particles in the
solution and HA titre of the unknown virus sample. Additionally, to examine the effect of
immunisation has on the concentration of antibodies and whether it has a positive
effect with producing antibodies against the virus.

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Materials & Methods:
Practical materials:
- 96-well microtitre plate

- Virus sample: Lec1 and Lec2

- 3% sRBC (Sheep Red Blood Cells)

- PBS (Phosphate Buffered Saline)

1. Transfer 50μL of PBS in each well of the two rows: A1-A12 and B1-B12 of a 96-well
microtitre plate.

2. Add 50μL of the ‘virus’ sample Lec1 to the first well of row A and mix thoroughly using
a pipette. Repeat this process until you reach well A11 and do not transfer to 50μL to
well 12 and discard from this well. Well A12 is used as a control.

3. Repeat step 2 for sample Lec2 using row B.

4. Once all the dilutions had been made, resuspend the sheep red blood cells (sRBC) by
inverting it several times. Add 50μL 3% sRBC to all wells from the highest dilution to
lowest dilution (12-1).

5. Mix the samples by gently tapping the plate and should suspend into the wells. After
completion of the titrations, label appropriately and set aside on the bench for 30
minutes.

6. From examining the plate for haemagglutination, if the reaction is negative, it shows
the results are valid. In contrary, if the PBS control is a positive haemagglutination result
presented, the results cannot be used and process should be repeated. A negative result
RBC precipitates to the bottom of the well, forming a distinct red dot in a cone-shaped
bottom. A positive result will form a mat on the base of the well where the RBC is
agglutinated by virus particle forming a lattice formation. The highest dilution reading
provides the agglutination of the RBC.

Results:
Below Figure 1 shows our example of representative results with both row A and B.
Both rows show a valid assay that allowed the titre to be determined.

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Figure 1: Haemagglutination titre of each sample (Row A and B) on 96-well microtitre plate

Virus calculation in virions per millimetre = 107 x HA titre

A (A9) = 107 x 1024 = 1.024 x 1010

B (B3) = 107 x 16 = 1.6 x 108

From figure 1, it shows my laboratory haemagglutination results, showing agglutinated

and non-agglutinated cells. From the results the two types of settling patterns are
visible. The wells that have not agglutinated are presented as dots, in this case the cells
have fallen at the bottom of the well. This indicates a negative agglutination reaction.
However, the cell which shows an even surface or red-carpet effect means the cells have
agglutinated, therefore this is known to be a positive agglutination reaction.

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Both row A and B contains the positive control sample which shows the presence of an
agglutination reaction as the serum contains specific antigens to the lectin antigens
which results in the attachment of RBCs that bind together. This indicates the virus is
present. The last positive well in row A is A9 which indicates the titre for this sample is
1024 (1/1024). Well 10-12 shows they had shown a non-agglutination reaction where
the cells fail to agglutinate. The last positive well in row B is B3 which indicates the titre
for this sample is 16 (1/16). In Row A, it contains a greater amount of virus sample than
Row B (A: 1.024 x 1010/B: 1.6 x 108).

Discussion:
In the experiment, there were 2 virus samples made into dilutions. This was successfully
done by carrying a dilution method. In the positive well, the binding of antigen was
successfully linked as these cells were agglutinated, this indicates the sample contains
specific antibodies to the lectin (virus) antigen allowing the RBCs and antigen to bind
together.

From our results, we observed that both Row A and B experienced agglutination at
different dilution factors. The reason for agglutination had in both A9 and B3 shows
both a positive result as sufficient antibodies are present in the sample to link the
antigens together. This forms a mast of antigen-antibody complexes on the bottom of
the well. Positive reaction can be analysed further using a haemagglutination inhibition
assay to measure the antibody levels in the solution (Virology blog, 2009). After this
point, the reaction is negative due to the depletion of antibodies present in the dilution
and presents no agglutination (Costabile, 2010). Row B has a titre lower than Row A
which may mean there are fewer antibodies present in the sample. Row A shows a HI
titer of 1024 which indicates at this dilution, this would be an antibody that is still
capable of recognising and binding to antigens on the virus. Higher titre values are
associated with greater antigenic properties and suggests that vaccination would
produce a greater immune response against the virus (Wikramaratna and Rambaut,
2015).

A limitation of our experiment, would be the manual method of analysing our HA or HI

results based on reading the plate and determining the titre values. This may induce
discrepancies in the assays within our results and subjective and since this is self-
recorded this makes it prone to human error and social desirability bias. To overcome
this, would be to alternatively use an enzyme-linked immunosorbent assay (ELISA) to
determine the HI titres (Jensen et al., 2013).

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Conclusion:
The haemagglutination assay is a simple technique that allows samples to be analysed
and assessed within a short period of time. Additionally, with the advantage of values
being read with a naked eye without the use of practical specialised equipment. The
results these experiment shows that haemagglutination assay is an essential tool as a
diagnostic armament against viruses. The assay allows to provide a quick results which
give attention to the management of patients. From our results, we observed
agglutination occurring in both row A and B at different dilution factors.

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References:
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3. Costabile, M., 2010. Determining the reactivity and titre of serum using a
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10. Wikramaratna, PS., Rambaut, A., 2015. Relationship between haemagglutination

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