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Agglutination tests

HA & HI

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Agglutination tests

Antibodies can agglutinate multivalentparticulate antigens, such as Red BloodCells (RBCs) or bacteria

Some viruses also have the ability toagglutinate with RBCs.

This behavior is called agglutination.

Serological tests based on agglutinationare usually more sensitive than thosebased on precipitation

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Examples

Slide Agglutination Test

Plate Agglutination Test

Tube Agglutination Test Passive Agglutination Test

Microscopic Agglutination Test

Haemagglutination test (HAT)

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Slide Agglutination Test

Used for serotyping (e.g. Salmonella)

Antigen: isolated Salmonella in suspension

Antibody: specific antisera against Salmonella

Place test Salmonella in a drop of saline on aslide

Add a drop of antiserum, mix and rock slide forapprox 1 minute

Examine for agglutination

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Slide Agglutination Test

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Tube Agglutination Test

Also known as the standard agglutination test or serumagglutination test (SAT)

Test serum is diluted in a series of tubes (doublingdilutions)

Constant defined amount of antigen is then added toeach tube and tubes incubated for ~20h @37C

Particular antigen clumps at the bottom of the test tube

Test is read at 50% agglutination

Quantitative Confirmatory test for ELISA reactors

Example: Brucellosis screening

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Tube Agglutination Test

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No agglutinationAgglutination

1/10 1/20 1/40 1/80 1/160 1/320 Neg. ctrl

In this case, the titre is 1/40

Tube Agglutination Test

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Passive Agglutination Test

Converting a precipitating test to anagglutinating test

Chemically link soluble antigen to inert

particles such as LATEX or RBC Addition of specific antibody will cause the

particles to agglutinate

Reverse PAT: antibody linked to LATEXe.g. Lancefield grouping in Streptococci.

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Quantitative MicroHemagglutination Test (HA)

Haemagglutination Test (HA)

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Haemagglutination

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Haemagglutination

RBC

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Viral Haemagglutination

Some viruses and microbes contain proteinswhich bind to erythrocytes (red blood cells)

causing them to clump together

NDV

Adenovirus III

AIV

IBV

Mycoplasma

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Viral Hemagglutination

the attachment of viral particles by their receptor sitesto more than 1 cell.

As more and more cells become attached in thismanner agglutination becomes visible

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Equivalence point:(suitable proportion between the virus particles and

RBCs)

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Negative control well (only RBCs+ buffer) (no

haemagglutinin)

Positive control well (contains haemagglutinin)

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Readings The results

Titer:The maximum dilution that givesvisible agglutination.

The end point:is the well with the lowestconcentration of the virus where there ishaemagglutination

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

haemagglutinating unit)

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Example of readings

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Titer = 32 HA units/ml

Hemagglutination test: method

1:8

1:2 1:21:21:21:2

8 16 32 64 128 256

virus

serial dilution

mix with redblood cells

side view

top view

One HA unit :minimum amount of virus that causescomplete agglutination of RBCs

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CALCULATIONS

For HI systems with different HA units:

(4 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.

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CALCULATIONS

If the titer is 32, How can you get 4 HA

units?

1/32 contain 1 HA

1/32 * 4 = 4/32 = 1/8.

1/8 dilution contain 4 HA units.

So 1/8 = (1/ 1+7) 10 wells * 50l/well = 500 l total volume.

500/8= 62.5 l virus + 437.5 HI buffer.

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WHAT DO WE NEED?

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PROCEDURE

(CONTROLs)

Always run four control rows:

_Positive:Contains antibodies against the specific virus

_Negative:Contains no antibodies against the specific virus

_ Antigen_ RBCs

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WASHING RBCs

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Why do we have to wash

RBCs?

To obtain pure RBCs and to get rid

from any other blood components

such as WBCs, immune complexes,and Abs

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Washing process

Take place 4-5 time .

Until get clear solution above the RBCs

after centrifugation .

Using PBS or normal saline .

Note :(avoid using water to wash RBCs

because it will definitely lead to RBCs lyses)

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Procedure

Obtain blood samples in tubes, spin at 1500RPM for 5 minutes.

Draw off the supernatant using Pasteur pipette.

Add 2ml PBS to each tube and move to a clean

test tube.

Centrifuge again. Each time draw off the

washing solution and add 10 ml PBS until thesolution above the RBCs layer becomes clear.

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Welldone !!

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HEMAGGLUTINATION

INHIBITION TEST (HI)

VIRUSE SERUM

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In the absence of anti-virus

antibodies

Erythrocytes

Virus

Virus agglutination of

erythrocytes

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In the presence of anti-virus

antibodies

Erythrocytes

Virus Anti-virusantibodies

Viruses unable to bind to

the erythrocytes

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Purpose: To quantitate serum antibody to a specificavian antigen

Procedure:1. A constant amount of haemagglutinating (HA) antigen is

added to each well in a microtiter plate.

2. The test serum is then placed in the first well andserially diluted.

3. The plates are incubated for one hour and then chickenRBCs are added to each well. If antibody is present inthe test serum the RBCs will not agglutinate with the HAantigen.

1. HI NEGATIVE wells will have a diffuse sheet of agglutinatedRBCs covering the bottom.

2. HI POSITIVE wells will have a well circumscribed button ofunagglutinated RBCs

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Antibody Titer

Is the lowestconcentration of

antibodies against

a particularantigen.

Figure 18.6

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Readings

The end pointis the well with the lowestconcentration of the serum where a clearbutton is seen.2 4 8 16 32 64 128 256 512 1024 2048 4096

The antibody titer in this row will be 512 (29).

(the lowest concentration of Abs which inhibit HA causedby the virus )