Quantitative Micro Hemagglutination Test

Quantitative(HA) Micro
Hemagglutination Test (HA)
 Definitions
• Agglutination: The clumping together of biologic
material, such as red blood cells or bacteria, that is
suspended in liquid.

• Hemagglutination: The agglutination of red

blood cells caused by an antibody either for red
blood cell antigens or for antigens that coat red blood
cells or by the presence of viruses or other
microbes.

• Hemagglutination test: sensitive test to measure

certain antigens, antibodies, or viruses, using their
ability to agglutinate certain erythrocytes.
hemagglutination
RBC
 Viral Hemagglutination
quantification of virus dilutions
• Many viruses contain proteins (e.g. virus
haemagglutination protein) which bind to
erythrocytes (red blood cells) causing them to
clump together i.e. agglutinate.
• Examples are:
• NDV
• Adenovirus III
• AIV
• IBV
 Viral Hemagglutination

• the attachment of viral particles by their receptor sites

to more than 1 cell.
• As more and more cells become attached in this
manner agglutination becomes visible
 Equivalence point:
(suitable proportion between the virus particles and
RBCs)
Negative control well (only RBCs+buffer) (no

Positive control well (contains haemagglutinin)

 Readings The results
• Titer: The maximum dilution that gives
visible agglutination.
• The end point: is the well with the lowest
concentration of the virus where there is
hemagglutination (no button),(HA1)
2 4 8 16 32 64 128 256 512 1024 2048 4096

The HA titer of this virus in this row is 256 or 28

(1:256 dilution contains (1 HA unit) (one
hemagglutinating unit)
Example of readings
 Hemagglutination test:
method
1:8

1:2 1:2 1:2 1:2 1:2

virus

serial
dilution
8 16 32 64 128 256
mix with red
blood cells

side view

top view

Titer = 32 HA units/ml

One HA unit :minimum amount of virus that will cause

complete agglutination of the red blood cells.
 CALCULATIONS

• For HI systems with different HA units:

(10 HA units for NDV ,8 HA unit for
AIV, 4 or 8 for adenovirus group III).

• divide the virus titer on the needed HA

unit.
 Hemagglutination assay: influenza virus

Hemagglutination assay. Seven different samples of influenza virus, numbered 1 through 7 at the left, were serially diluted as indicated at
the top, mixed with chicken red blood cells (RBC), and incubated on ice for 1 to 2 hours. Wells in the bottom row contain no virus.
Agglutinated RBCs coat wells evenly, in contrast to nonagglutinated cells, which form a distinct button at the bottom of the well. The HA titer,
shown at the right, is the last dilution that shows complete hemagglutination activity. (From Fields Vriology (2007) 5th edition, Knipe, DM &
Howley, PM, eds, Wolters Kluwer/Lippincott Williams & Wilkins, Philadelphia Fig. 2.9)
WHAT WE NEED ?
 HEMAGGLUTINATION
INHIBITION TEST (HI)

VIRUSE SERUM
 hemagglutination inhibition
(HI) test
• an assay for the presence of specific antiviral
antibodies in a test serum.
• convenient and commonly used assay that
requires cheap reagents and is read by eye.
• the antibodies bind to the haemagglutinin
protein in the envelope of the virus. This
blocks the haemagglutinin protein from binding
with the receptor site on chicken RBCs
• The highest dilution of serum that inhibits
hemagglutination is the HI titer of the serum.
In the absence of anti-virus
antibodies

Erythrocytes

Virus
Virus agglutination of erythrocytes
In the presence of anti-virus
antibodies

Erythrocytes

Virus Anti-virus
antibodies

Viruses unable to bind to

the erythrocytes
 Antibody Titer
• Is the lowest
concentration of
antibodies against
a particular
antigen.
 Readings
• The end point is the well with the lowest
concentration of the serum where a clear
button is seen.
2 4 8 16 32 64 128 256 512 1024 2048 4096
 PROCEDURE
(CONTROL)

• Always run four control rows:

_ Positive antiserum row (Contains antibodies
against the specific virus)
_ Negative antiserum row (Contains no antibodies
against the specific virus)
_ Antigen row.
_ RBCs row.
 The commonly-used HI tests in chickens

• for Newcastle disease (Paramyxovirus-1), Infectious bronchitis

(Coronavirus), and EDS-76 (adeno-virus),HI tests may also be
carried out for Avian Influenza.
WASHING THE RBCs
 Why we have to wash them ?

• To obtain the RBCs and get rid from

any other blood components such as
WBCs, immune complexes, and Abs
 Washing process

• Take place 4-5 time .

• Until get clear solution above the RBCs
after centrifugation .
• Using PBS or normal saline .

Note :(avoid using the water as washing

material cause it will definitely lead to the RBCs
lyses )
 Procedure
• Fill the blood samples in tubes ,centrifugation at
1500 round/min for 10 min .
• Draw off the plasma and WBCs coat using
pipette filter spelling it into the sink .
• Add the PBS to each tube and centrifuge again,
each time draw off the washing solution and
add new amount until we get a clear soultion
above the RBCs layer ,almost 3 times .
THANK YOU