Haemagglutination Inhibition Assay Report: Biol 2402, Viral Infections

This report details a Haemagglutination Inhibition (HI) Assay experiment, conducted to determine the inability of a virus to agglutinate red blood cells (RBCs). The experiment aimed to measure the amount of antibodies present in a serum sample against the influenza virus. The methodology involved preparing serum dilutions, reacting them with a fixed amount of virus, and then adding RBCs. The results, including pre- and post-vaccination serum antibody titers, are presented and discussed. The report explains the principles of the HI assay, its advantages over other assays, and the interpretation of the results. The discussion section includes the calculation of the antibody titer and its significance in indicating the presence of antibodies. The report also highlights the importance of controls and proper serial dilutions for accurate results and concludes with the test's application in virus assays and the future scope of research. The report is based on a practical experiment from a Biol 2402 Viral Infections course.

Running head: HAEMAGGULINATION INHIBITION ASSAY
HAEMAGGLUTINATION INHIBITION ASSAY
Name of the Student
Name of the University
Author note

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Table of Contents
Aim of the experiment.....................................................................................................................2
Introduction......................................................................................................................................2
Method.............................................................................................................................................3
The materials required for 4 students..........................................................................................3
The pre vaccination sera test........................................................................................................3
The post vaccination sera test......................................................................................................4
The back titration while working on the viruses.........................................................................4
Result...............................................................................................................................................5
Discussion........................................................................................................................................6
Conclusion.......................................................................................................................................7
References........................................................................................................................................8

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Aim of the experiment
The aim of this experiment is determining the inability of virus to agglutinate the red
blood cells (RBCs) by doing haemagglutination inhibition assay.
Introduction
The haemagglutination inhibition test is one of the simplest, very sensitive and
inexpensive and very rapid and so it has become one of the method which is chosen in the
practical purposes for doing the assessment of the influenza viruses. The test is dependent on the
haemagglutination of the influenza virus and the specificity of the antibodies to bind to the virus
to inhibit from agglutination. The ability of the influenza virus to agglutinate the red blood cells
can get exploited further for measuring the amount of antibody to the influenza viruses present in
a sample of serum. If antibody is present in the serum sample then, then the antibodies will
attach to the viruses and will prevent the virus from the red blood cells getting agglutinated. (2).
Dilutions of the serum are prepared and reacted with the fixed number of viruses and then the red
blood cells are given. The suspension of the viruses consists of the fixed number of viruses and
the amount of the virus must be such that it can easily agglutinate the red blood cells. The fixed
number of viruses must contain 4HAU per 25 μl. This value is the standard or the convention for
doing the assay (1).
The haemagglutination inhibition assay is mostly used for measuring the amount and
mainly against influenza virus. One of the example of the viral proteins having the capability to
bind to the red blood cells is the haemagglutinin proteins of the influenza virus. Thus protein
have the capability to bind to the residues of the sialic acid on the surfaces of the cells (3). It is to
be determined that how much the virus can be diluted before the agglutination property of the

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virus get cancelled. The last of this assignment is that at the end the last well of the titre must
show complete haemagglutination . The titre value of HA is determined as the reciprocal of the
highest dilution which is causing the incomplete haemagglutination (4). Both the agglutination
and agglutination inhibition assay agglutinate the red blood cells but the main difference between
the haemagglutination test and the haemagglutination inhibition assay is that the normal
haemagglutination inhibition assay cannot differentiate between the virus particles which are
very infectious and the virus particles which can degraded and which do not have the capability
to infect cells.
Method
The materials that are used in the experiment includes 96 well microtitre plate (the pre
vaccination serum of 1:10 dilution), 0.5 % Chicken red blood cells (the post vaccination serum
of 1:10 dilution), the suspension of the influenza virus (25μl and 50μl respectively20 ml PBS
(25μl and 50μl tips) (7).
The pre vaccination sera test
At first, 25 μL of PBS should be placed in the wells of 2 to 11 and then 50μl of the
serum, pre vaccination, should be placed to well number 1. By adjusting the 25μ pipette, the
serial dilutions was performed from the well number 1 to the well number 11 and 25μl must be
discarded from the last latter. Next, 25μl of the virus solution with which the working is done
should be given to the wells from 1 to 11. After that it was mixed and incubated at the normal
temperature for almost about 30 minutes. (7).

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The post vaccination sera test
25μl of PBS was placed in the wells 1 to 11 and also another 25μl in the control wells.
Then 50μl of the post vaccination serum should be given to the well number 1. Then by using the
pipette, the serial dilution was performed from the well number 1 to the well number 11and the
last 25μl should be discarded. Then, 25μl of the solution with which we are working should be
given to the wells from 1 to 11. Then it should be mixed and incubation must be done for another
30 minutes. (11).
The back titration while working on the viruses
25μl of the PBS must be added from well 2 to the well 12. Secondly, 50 μl of the working
virus should be placed to the well number 1. Then serial dilutions was executed from the well
number 1 to the well number 11 and then 25 μl was discarded from the last well. Then PBS of
25μl is added to the wells from 2 to 11. Next, the whole solution must be mixed properly and
incubated for 30 minutes. The after 30 minutes are over, the red blood cell suspension from the
chicken must be mixed properly and then 50μl of 0.5% should be given to all the wells. Then
mixing and incubation was done at room temperature. The time for the control to the button was
noted. The end point of the dilution is considered as the highest dilution of the serum causing the
inhibition of the agglutination completely. The HI titre is the reciprocal of the end point (12).
Result
Result of the haemagglutination inhibition assay

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The First column in this picture shows the dilution of serum of the serum of pre vaccination, the
second row is showing the dilution of the serum for the post vaccination and the third row shows
the dilution of the virus solution for the back titration.
Discussion
Yes, the results that are obtained from the experiment is according to the outcome that are
predicted. The value of the antibody titre from the pre vaccination is 0 and the value of antibody
titre from the posy vaccination is 80. In the picture of the result it is seen that in the control wells
agglutination has occurred and also in all the wells of the virus solutions but in the wells of the
post vaccination serum the agglutination has stopped occurring at the titre value 1/80. The titre
value is the ratio which is used for explaining the amount of some substances present is a
solution. In the health issues, the titre value represents the number of antibodies that are present
in the blood of the person. The antibodies are proteins which are found to circulate continuously
in the blood. So after giving vaccination against a particular disease the number of antibodies in

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the blood increases. So the titre value gets increased after giving vaccination against a particular
diseases. In this case also the titre value has increased because of the increase in the amount of
antibody. The back titration is done to determine the amount or concentration of the unknown
variable, in this case it is antibodies and this is done by finding the amount of compound that is
remaining with a known concentration. The haemagglutination inhibition assay is more
advantageous than the haemagglutination assay because the haemagglutination test vary
according to the protocols and the expression of the end points (8). The variation of the test
limits the comparison between the different strains of influenza. Everything went well while
doing the experiment and it was quite easy to use. The control is used in any experiment to
differentiate between the original results obtained from the result. If no control sample is there,
then the original result that is obtained will not be differentiated. The differences between the HI
assay and the HA assay are more variations are there in the HA assay in comparison to HI assay
and also in the normal haemagglutination test differentiation cannot be done in between the
infectious virus particles and the also the virus particles which cannot be differentiated and
cannot infect cells. The results of the haemagglutination assay test vary from one laboratory to
other. Haemagglutination assay can be applied to the viruses which can agglutinate RBCs and
cannot be applied to any other viruses who do not have the property to agglutinate the red blood
cells (9). For the medical purposes, the haemagglutination tests can be applied in the preparation
of vaccines against the disease influenza. The haemagglutination inhibition test is used in
laboratories for blood typing test and in doing the viral assay. One of the important disadvantage
of the haemagglutination inhibition test is the serial dilution must be done properly otherwise the
titre value will get changed (10). If improper titre value comes then the amount of antibodies
required to immune the person will get altered.

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Conclusion
The haemagglutination inhibition test is mainly used in virus assays whose structures
contain proteins like the influenza virus. The titre value obtained from this test depicts the
antibody required to prevent the virus from forming agglutination. Presently in the medical field,
this test is used in the detection and identification of the influenza viruses but in future much
researches should be done on this test so that this test can be used in the assessment of other
viruses.

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