AGGLUTINATION

Agglutination:
• Is the clumping of antibody–antigen complex to form insoluble and
visible aggregates.
The word agglutination comes from the Latin agglutinare, meaning "to glue to."

Antigen:

Antigen is any molecule that binds specifically to an antibody.

Antibody:

Antibodies (immunoglobulins) are proteins secreted by the immune system

to identify and neutralize foreign objects (antigens), such as bacteria and
viruses.

Factors affecting the agglutination reaction in vitro:

1) Antigen to antibody ratio:

• The ratio between antigen and antibody influences the detection of

antigen-antibody complexes.
• Antigen or antibody excess make invisible reaction.

Prozone phenomenon (antibody excess):

• There are too many antibodies.

• Antibodies saturating all antigen sites
• No antibodies forming cross-linkages between cells or particles
so, no agglutination appears (false-negative reactions).
Zone of equivalence:
• Antibodies and antigens are present in an optimum ratio.
• This leads to cross-linkages between acells or particles, so
agglutination appers (positive reaction).

Post-zone phenomenon (Antigen excess):

• There are too many antigens
• Any agglutination is hidden by masses of unagglutinated
antigens that gives false-negative reactions.
 These false-negative reactions can be detected by repeating the
test at a higher dilution of sample, which reduces the antigen or
antibody concentration into the range that produces visible
agglutination.

Number of Antigen Sites:

• The more antigen sites on a cell result in more cross-linkages between

cells.
• These cross-linkages result in more visible agglutination.
Size and Structure of the Antibody:
• Larger antibody causes more cross-linkages between different cells.
• The larger antibodies (IgM) can reach between more antigen sites on
different cells and therefore causing stronger agglutination reactions.
• IgM antibodies also have more binding sites to react with antigens and
potentially causing cross-linkages between 5 different cells.
Distance between cells:
• The small IgG antibodies usually cannot reach between two cells as

the distance between its two arms is narrow.

• The larger antibodies, IgM, can reach between cells that are further
apart and cause agglutination.
• Centrifugation of the cells attempts to bring the cells closer together,
so enhance agglutination.

2) Cell surfce electric charge (Zeta potential):

• Zeta potential is an electric charge surrounding the red blood cells

makes the cells maintain a certain distance from each other.

 • It makes repulsion between the red blood cells when suspended in
saline.
• It is cause by sialic acid groups on the red blood cell membrane which
gives the cells a negative charge.
• The positive ions in saline attracted to the negatively charged red
blood cells.
• The net positive charge surrounding cells in saline keeps them far
apart due to repulsion from electric charges.
• Smaller antibodies (IgG) cannot cause agglutination when zeta
potential exists
• To overcome zeta potential techniques add albumin to test mixture as
OH- groups of albumin neutralize positive charge.

Classification of agglutination reactions:

1. Direct agglutination reactions:
RBC (Ag) + specific Ab = agglutination
Examples:
• ABO blood group typing
 Rh (D) Ag

2. Passive (indirect) agglutination

Patient serum, Ab (IgM) + soluble Ag adsorbed particle =
agglutination
 Passive Agglutination is a very sensitive method for antibody
detection.
 RBC, bacterial cells or inert particles such latex can be used as
a carrier for soluble antigens.
 The soluble antigen is absorbed onto the carrier cell or particle,
then reacted with the specific antibody.
 This makes the reaction more visible than precipitation.
Example:
o Rheumatoid factor detection:
Rheumatoid factor is IgM-anti IgG which agglutinates
latex beads coated with IgG.
o C-Reactive Protein detection (CRP):

CRP is an immunologic reaction between serum

containing CRP (antigen) and the corresponding antibody
coated on latex particles.

Test procedure:
Add 1 drop CRP latex to 1 drop of patient serum (may be diluted)
 then stir & mix.

Grades of the positive reaction:

 4+ Large clumping with clear fluid in the background
 3+ Moderate clumping with fairly clear fluid in the
background
 2+ Small clumping with a slightly opaque fluid in the
background
 1+ Very small clumping with fluid definitely opaque

Practical work sheet

Give an account on:
Factors Affecting agglutination tests in vitro:

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Types of agglutination test and write an example for each:

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Procedure of CRP detection:

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Direct Antiglobulin Test (DAT)

“Direct Coombs’ test

Aim:
To detect the coated red blood cells with incomplete immunoglobulin (IgG) in
vivo.

Principle:

• IgG + RBC = give complex with no agglutination (in vivo).

• Add anti human globulin “IgM” to the complex = agglutination (in
vitro)

Sample:
• Blood drawn into EDTA is preferred but oxalated or citrated blood may
be used.
• The blood sample should be tested as soon as possible after collection
and should not be stored.

Reagents, Equipment and Supplies:

• Polyspecific AHG (Coombs)

 • Isotonic saline
• Coombs Control Cells
• Pipettes
• 12 x 75 mm tubes
• Centrifuge
• Microscope

Procedure:

1. Put 1-2 drops of blood in EDTA tube.

2. Wash this tube three times with isotonic saline.
3. Prepare a 5-10% suspension from the washed cells.

4. Add one drop of the washed 5-10% suspension to a tube.

5. Then, add one drop Polyspecific Antihuman Globulin and shake to mix.

6. Centrifuge the tube for 30 seconds.

7. Immediately resuspend gently and examine for agglutination.

Interpretation:

1. No agglutination indicates a negative DAT - nothing is coating the

patient's cells in vivo.
2. Agglutination with Polyspecific AHG indicates a positive DAT, due to
either IgG antibodies or complement coating the patient's cells.

Preccautions:

1. The washing step must proceed uninterrupted. A delay in washing may

cause antibodies to be eluted off cells and washed away.

2. AHG must be added to the cells immediately following washing.
 Antibodies may elute from the cells if they are allowed to sit in saline
without the addition of AHG.
3. The Coombs Control Cells must be positive. Ther reasons for false

negative test include:

 Inadequate washing of the cells. Residual serum neutralizes the AHG

so that it cannot react with the antibody-coated Coombs Control
Cells at the end of the procedure.

Be sure the cells are thoroughly resuspended between each wash.

 A delay in adding AHG. Antibodies that were attached to the red

cells may elute off into the saline, and neutralize the AHG when it is
added.

Add the AHG immediately after the final wash.

 AHG was omitted “inactive” or was diluted out by too much residual
saline in the tube.

Drain the last wash well and blot before adding AHG.
 Practical work sheet
Give an account on:
The aim of direct Coombs’ test:

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Procedure of direct antiglobin test:

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