[Authentic] HAEMAGGLUTINATION INHIBITION

EXPERIMENT 6: HAEMAGGLUTINATION INHIBITION ASSAY (HI)
The ability of influenza to agglutinate RBC can be exploited further to measure the amount of antibody to influenza in a serum sample. If there is antibody present, it will bind to the virus and prevents the virus from agglutinating RBC. We call this haemagglutination inhibition.
A series of dilutions of serum are reacted with a fixed amount of virus, and then RBC is added. The virus suspension is a fixed amount and it is an amount that would normally agglutinate the RBC.
How far can we dilute our serum before it no longer inhibits agglutination?
How do we get a preparation that contains 4 HAU?
We carry out a HA. If the HA titre is 640:
The well containing virus at the dilution of 1/640 had 1 HAU/50 μL.
We want 4 HAU. Divide 640 by 4 to get 160.
This means that a 1/160 dilution contains 4 HAU/50 μL.
But, in our HI assay, we only use 25 μL of virus, so if we used this, we would only have 2 HAU/25 μL. So we must divide the 1/160 by 2 giving us a dilution of 1/80.
So, to get 4 HAU/25 μL, we must divide our HA titre by 8.
The HI test could be used for virus identification, to determine the antigenic relationship of different virus isolates, and for the identification and quantification of viral antibody to virus. This assay is used to monitor the antigenic variation of influenza A viruses.
In the lab, the HI assay is often performed using both acute and convalescent serum. In the vaccination situation, we can take a pre-vaccination blood sample and another post vaccination sample 4 weeks later. Using these two samples we can observe the rise in antibody titre. For influenza, if we obtain a ≥ 4-fold increase in antibody we say that the person has seroconverted. In a group of people, we can calculate an average titre, known as the geometric mean titre (GMT).
Serum must be treated as this removes it of non-specific inhibitors.
Materials (per 4 students) 96-well V-bottom microtitre plate
1:10 diluted pre vaccination serum
0.5% chicken RBCs
1:10 diluted post vaccination serum
Influenza virus suspension (4 HAU per 25 μL)
25μL and 50μL pipettes
20ml PBS
25μL and 50μL tips
You will require 3 rows in a microtitre plate. See plate example Page 63.
1. Pre-vaccination sera test
2. Post-vaccination sera test
3. Virus dilution
Pre-vaccination sera test Figure 9.1
1. Aliquot 25μL of PBS in wells 2-11, and 25μL in the control wells.
2. Add 50μL of pre vaccination serum to well 1.
3. Using the25 μL pipette, perform serial dilutions from well 1 through to well 11. Discard last 25μL.
4. Add 25μL of working dilution virus (4 HA units/25 μL) to wells 1-11.
5. Mix and incubate at room temperature for 30 minutes.
Post-vaccination sera test Figure 9.2
1. Aliquot 25 μL of PBS in wells 2-11, and 25μL in the control wells.
2. Add 50 μL of post vaccination serum to well 1.
3. Using the 25 μL pipette, perform serial dilutions from well 1 through to well 11. Discard last 25μL
4. Add 25 μL of working dilution virus (4 HA units/25 μL) to wells 1-11.
5. Mix and incubate at room temperature for 30 minutes.
Method: Back titration on the working dilution of the virus:
1. Add 25 μL of PBS to wells 2-11
2. Add 25μL of PBS to well 12
3. Add 50 μL of working dilution virus to well 1.
6. Using the 25 μL pipette, perform serial dilutions from well 1 through to well 11. Discard last 25μL
7. Add 25 μL of PBS to wells 2-11 (this is to equate volumes in the backtitration and your HI test)
8. Mix and incubate at room temperature for 30 minutes
Then
9. After 30 mins has elapsed gently mix chicken red blood cell suspension to ensure cells are uniformly suspended. Add 50 μL of 0.5% chicken red blood cells to all wells.
10. Mix and incubate at room temperature. Note time for controls to button (about 30 minutes). Draw the pattern of each well. (Examples on page 64). Record end point of your sample.
The end point is regarded as the highest serum dilution causing complete inhibition of agglutination (note: streaming of RBCs occurs when the plate is tilted at 450).
The HI titre is the reciprocal of the end point (i.e. HI titre = 1/End point).
RESULTS:
HAEMAGGLUTINATION INHIBITION ASSAY (HI)
VIRUS DILUTION FOR BACK TITRATION
SERUM DILUTION FOR POST-VACCINATION SERUM
SERUM DILUTION FOR PRE-VACCINATION SERUM
ANTIBODY TITER FROM PRE-VACCINATION: 0
ANTIBODY TITRE FROM POST-VACCINATION: 80
GOOD RESOURCE:
http://bit.ly/2W5Lr38
ALSO INCLUDE:
Why is there a greater titre in Post-Vaccination?
For what is Back Titration?
For what is the Control? Why is it important that the assay must be properly working/functioning?
Why is Haemagglutination Inhibition(HI) Assay better than Haemag…
 

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