Sensitization and Agglutination

Antigen-Antibody reaction

• Red cell Ag-Ab reaction can be detected by a number of techniques

• Most frequently used – Hemolysis

• Occurs if the entire complement sequence is activated following Ag-Ab interaction

• Frequently stops at C3 – no lysis• In vitro, lysis of RBC’s rarely occurs.

– Agglutination

• Used as indicator of Ag-Ab reaction

Sensitization

• Sensitization is whereby the Ab binds to Ag on the surface of the cell.

• Sensitization does not always result in visible agglutination.

• IgG is too small to span the distance between two red cells.

• So IgG does not always result in visible agglutination.

Agglutination

• Agglutination occurs when bound Ab links

adacent red cells forming clumps.

• Red cells must be close enough for the

Fab portion of Ab to bind and make

bridges between cells.

• IgM can easily induce agglutination.

Agglutination Reactions• Two Stage Process:

– Stage 1 Sensitization: • attachment of Antibody to Antigen on the RBC

membrane.

– Stage 2 Lattice formation (agglutination): • formation of bridges between the sensitized red

cells to form the lattice that constitutes agglutination.

This represents what occurs during stage one of agglutination.

• Antibody molecules attach to their corresponding antigenic site (epitope) on the red blood cell membrane.

• There is no visible clumping.

• Red cells must be close enough for the Fab portion of Ab to bind and make bridges between cells

Stage 1: Sensitization

This represents what occurs during stage 2 of agglutination:

Antibody molecules crosslink RBCs forming a lattice that results in visible clumping or agglutination.

Stage 2: Lattice Formation

The Zeta Potential

• The electric repulsion between cells

• This explains why cells do not agglutinate

• Red cells have negative charge due to

sialic acid molecules

• When red cells are in solution containing free ions:– Cations are attracted to the –vely charged red cells– This forms a repelling cloud around the cell

• The Zeta Potential can be varied by altering the charge on red cells

• This can affect both sensitization and agglutination

• Reducing the cloud density allow Abs to approach the cells, sensitize and then agglutinate them

Factors affecting the Zeta Potential

1- Using Enzyme

2. Introduction of bipolar Albumin

• Albumin dissipates some of the +ve charges around cells, reducing zeta potential

Albumin

Factors affecting the Zeta Potential

3. Increase the ionic strength of the medium• Increasing conc. of cations in medium cause

– Increase in the density of ions around the red cell which cause

• Size of cloud of cations is decreased

• Zeta potential decreases

• Red cell approach each other easily

• Agglutination is facilitated

Increase the ionic strength of the medium

Factors affecting the Zeta Potential

4. Decreasing the ionic strength of medium by using low ionic strength saline (LISS)

– Decreasing conc. of cations in medium

– Leads to decrease in density of ions around red cells

– This increases sensitization

– But decreases agglutination

Decreasing the ionic strength of medium by using low ionic strength saline (LISS)

Factors affecting Red Cell Sensitization

1- Ratio of Ab to Ag– Sensitization occurs easily when at higher conc. of Ab– This can be done by increasing conc. of serum

containing the Ab to conc. of cells

Factors affecting Red Cell Sensitization

2- The pH of reaction mixture– At a pH below the

isoelectric point, Abs have +ve charges

– This makes it easier for the Ab to bind to the –vely charged red cells

– Optimal pH for sensitization is 6.5 to 7.5 (Ab +vely charged)

Factors affecting Red Cell Sensitization

3- Temperature– Ag- Ab reactions are

exothermic – Therefore, Abs bind to

a greater degree at lower temperature

– But at lower temperatures, rate of reaction is reduced

– To speed up reaction, tests are done at 37oC

• Temperature can also affect Ag accessibility on red cells – Some IgM Abs bind best at 4oC (cold Abs)

• Temperature can make conformational changes in the Ag– More Ag sites are exposed as the temperature is

lowered allowing increased binding of Ab

• Most naturally occurring cold Abs are of no clinical significance– Compatibility testing is done at 37oC

4oC 37oC

Factors affecting Red Cell Sensitization

4- Ionic strength of the medium– When RBCs are suspended in LISS the cloud

of ions around the cell is less dense than in isotonic saline

– Reduced conc. of cations surrounding RBCs allow +vely charged Abs easier to access Ag sites

– Rate of sensitization increases

Factors Influencing RBCs Agglutination

• Agglutination occurs when RBCs are close enough allowing the Ab to bridge adjacent cells

Factors Influencing RBCs Agglutination

1- Ionic strength of Medium– Rate of sensitization increases in LISS

BUT:– Agglutination is impaired because of increase

in zeta potential

• (Rule: reduced zeta potential facilitate agglutination)

Factors Influencing RBCs Agglutination

2- Presence of Albumin in medium

3- Enzyme treatment of red cells• Remove –vely charged sialic acid• & therefore reduce the zeta potential• This make cells come closer & agglutinate• But, certain Ags can be destroyed by enzymes (M,

N, S, Fya, Fyb)

4- Temperature (previously discussed)

Factors Influencing RBCs Agglutination

5- Antigen Density– The greater the number of

Ags on red cell, the greater the sensitization

• Binding of +vely charged Abs to red cells lower the zeta potential

• And therefore enhances agglutination

– Increased Ag density also increases chance of bridging

Factors Influencing RBCs Agglutination

6- Ag Clustering and Mobility – Clustering facilitates agglutination by

increasing likelihood of Ab binding at that site– Cluster of some Ags can occur after enzyme

treatment of cells

Clustering of Ags

Factors Influencing RBCs Agglutination

7- Antibody Characteristics– Ability of Ab to agglutinate cells depend on the Ig

class– IgM has a wider span than IgG, and therefore more

effective agglutination– IgG can be chemically modified to increase its span

IgMAg

300 Ao

IgG IgG

150 Ao250-300 Ao

7- Antibody Characteristics

Summary

Sens. Agg.

Removal of RBCs sialic acid by enzymes

Introduction of bipolar albumin ___

Increasing ionic strength of the medium

decreasing ionic strength of the medium