1. Veterinary Medicine /Poultry Disease (Practical subjects) / HI/ 4 stage / Dr.Nawar 2014-2051
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*Haemagglutination test (HA):-
Definition: It is ability of certain viruses (haemagglutinating viruses) to aggregate
RBC in Suspension.
*Principle of Haemagglutination test (HA):-
1-Ability of Certain Virus to agglutinate RBC due to the binding of
Haemagglutinin-Neuraminidase (HN) Protein of virus to Receptors on the
surface of RBCS.
2-The Certain Virus that can agglutinate RBC are (Paramyxovirus,
Orthomyxovirus, Reovirus, Adenovirus, togavirus) and Mycoplasma.
3-The agglutinate appear as precipitate of RBC in the well of the plate.
*Material of Haemagglutination test (HA):-
1-HA plate (Macrotiterplate or Microtiterplate plate).
2-Macropipete & Micropipete.
3-Chemical agent such as PBS (Phosphate buffer solution) or Normal Saline and
EDTA.
4-Biological agent such as washed RBC (the blood sample was taken from Sheep,
Guinia pig, Chicken, Rabbit, Human).
5-Virus which taken from vaccine ampule or infected Chicken.
RBC (washed) + HA Virus Setting Pattern
2. Veterinary Medicine /Poultry Disease (Practical subjects) / HI/ 4 stage / Dr.Nawar 2014-2051
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*Procedure of Haemagglutination test (HA):-
1-Add (0.5 ml) Normal Saline to all well.
2-Add (0.5 ml) of the Virus to the first well and mixed and then transfer by
Micropipete to the next well and third well,…etc.(Make serial dilution 2 fold) and
finally 0.5ml discard.
3-Add the (washed RBC 1%) to each well.
4-Make the Two Control well by adding blood to each well then put the virus in
the one well and normal saline on the others.
Control
موجبةنتيجة اعطى تخيف أعلى مقلوب =الفايروس معيار×2
0.5 ml N.S
0.5 ml Virus
0.5 ml RBC
͟͟͟͟͟͟͟͟͟
͟͟͟͟͟͟͟͟͟1
2
HA2HA4HA
͟͟͟͟͟͟͟͟͟1
4
͟͟͟͟͟͟͟͟͟1
8
1
16
1
32
1
64
1
128
1
256
+Ve -Ve
0.5ml RBC
+
0.5 ml Virus
0.5 ml RBC
+
0.5 N.S
3. Veterinary Medicine /Poultry Disease (Practical subjects) / HI/ 4 stage / Dr.Nawar 2014-2051
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. (HA unit تالزن وحدة. )128 = 2 × 64=Titer
5-Mixed by tapping the plate gently, then allow RBC to settle for about
(30 minute) at room temperature.
6- Haemagglutination is observing by the presence or absence of tear (net) shape of
RBC.
7-The Titeration should be read to highes dilutuion giving complete HA.
8-Method of Wash RBC:-
Take (1ml) of blood with (EDTA in tube), if 1ml blood can put (2ml) of Distal
Water (1blood:2D.W) in centrifuge can result RBC, Buffycoat, Plasma by pipete
can draw the plasma and buffycoat content and repeat the method for 3 times to get
RBC washed.
9-The concentration of wash RBC (0.5-1%).
1ml of RBC+99ml of N.S=1%.
If u want 0.5% :-
V1×C1=V2×C2
1%×100ml=V2×0.5%
V2= =200 ml
0.5 ml RBC+199.5 ml of N.S=0.5%.
10-Do not read the test after (40minute) because the agglutination disappear
because the elusion by Neuramindase protein.
*The Aim (Uses) of the Haemagglutination (HA) test:-
1-Titeration the virus.
2-Measuringe of 4 HA unit.
3-Diagnosis the virus which have the ability to agglutination of RBC.
1×100
0.5
4. Veterinary Medicine /Poultry Disease (Practical subjects) / HI/ 4 stage / Dr.Nawar 2014-2051
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*Haemagglutination Inhibition test (HI):-
(Serogical test)
*Definition: It is a serological test depending upon antigen antibody reaction in
which inhibition of Haemagglutination occurs due to masking of virus receptors
by specific Antibodies.
*Principle of Haemagglutination Inhibition test (HI):-
Specific attachment of Antibody to Antigen sites (on HA molecule of virus)
interfere with binding between the virus (Haemagglutinate) and receptors on the
RBC.
*The Uses of the Haemagglutination Inhibition (HI) test:-
1-Titeration of antibody.
2-Evaluation of Vaccine.
3-Typing of Haeagglutinating Virus (using antisera) e.g influenza.
RBC + HA Virus + Antibody Haemagglutination Inhibition
+ +
5. Veterinary Medicine /Poultry Disease (Practical subjects) / HI/ 4 stage / Dr.Nawar 2014-2051
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*Procedure of Haemagglutination Inhibition (HI) test:-
1-Add (0.5 ml) of Normal Saline to each well U-bottomed of Macrotiterplate.
2-Add (0.5 ml) Serum (Abs) in the first well of plate then make serial dilution
(Add to first well and mixed and then transfer to next well and third well…etc) and
finally discard 0.5ml.
3-Add (0.5 ml) of 4HAU to each well.
4-Leave for (30 minute) at room temperature.
5-Add (0.5ml) of Washed RBC to each well.
6-Mixed gently then allow RBC to settle for about (30 minute).
1024=16×64 =
قوي الفايروس ان ايجدأ.
Control
0.5 ml N.S
0.5 ml Serum
0.5 ml 4HAU
0.5 ml RBC
1
2
1
4
1
8
1
16
1
32
1
64
1
128
1
256
موجبةنتيجة اعلى مقلوب = الفايروس معيار× (Titer)4HA
Virus
+RBC
-Ve
N.S
+RBC
+Ve
Serum
+RBC
+Ve
