Slide 1 of 52

This educational material has been

produced by the London and South East
Joint Technical Advisory Groups
Transfusion Training Committee.
It is presented on an as is basis to assist
in training. Please feel free to use or
modify as required.
We positively welcome constructive feedback.
Please contact Malcolm Needs on 020-8258-8346 or
malcolm@needs.nbs.nhs.uk.

The London and South East Joint Technical

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Those slides marked with

a red asterisk* are
considered essential
knowledge by the
Transfusion Training
Committee. Others may
be thought of as the
icing on the cake!
The London and South East Joint Technical

Slide 3 of 52

Antibody/Antig
en Reactions.
The London and South East Joint Technical

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An Antigen.

An antigen can be defined as a

substance that, when
introduced into the circulation
of an individual lacking that
antigen, can stimulate the
production of a specific
antibody.
The London and South East Joint Technical

Slide 5 of 52

An Antigen.

The antigenic determinant,

which is more correctly termed
an epitope, is the antibodybinding portion of the antigen,
which is in the region that is
complementary to the
combining site of the antibody.
The London and South East Joint Technical

Slide 6 of 52

An Antibody.

An antibody can be defined as a

serum protein (i.e. an
immunoglobulin with specific antigen
binding sites) produced as a result of
the introduction of a foreign antigen,
that has the ability to combine with
(and, in many cases, destroy) the
cells carrying the antigen that
stimulated its production.
The London and South East Joint Technical

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The Law of Mass

Action
governs
antibody/antigen
reactions.
The London and South East Joint Technical

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[Ag] + [Ab]

k1
k2

[AgAb]

The London and South East Joint Technical

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[Ag] + [Ab]

k1
k2

[AgAb]

The London and South East Joint Technical

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Reactions Are Observed in

Three Ways in the
Laboratory.

Agglutination.

Haemolysis.

Inhibition.

The London and South East Joint Technical

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The Forces That Enable

Antibodies to Bind to
Antigens Include:

Ionic Bonds.

Electrostatic attraction of positive

and negative charges.

Hydrogen Bonds.

Weak bonds that form between a

proton donor or proton acceptor on
the antigen and the opposite amino
acids on the antibody. These two
groups share a hydrogen atom.
The London and South East Joint Technical

Slide 12 of 52

The Forces That Enable

Antibodies to Bind to
Antigens Include:

van der Waals forces.

Polarisation of external clouds of

electrons in an atom. When the
electrons that spin about the
nucleus of an atom swing to one
side, a positive charge is created on
the opposite side. This temporary
positive charge attracts the
electrons of a nearby atom.
The London and South East Joint Technical

Slide 13 of 52

The Forces That Enable

Antibodies to Bind to
Antigens Include:

Changes to the ordered

hydration of the membrane.

Stabilising antigen/antibody
complexes by the release of H2O,
causing hydrophobic amino acids to
associate more closely.
Reduction in membrane H2O enhances
the ability of fat-soluble
immunoglobulin to associate with
some antigens.

The London and South East Joint Technical

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Factors
Affecting
Antibody/Antige
n Reactions.
The London and South East Joint Technical

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Factors Affecting
Antibody/Antigen Reactions.
Temperature.
Incubation time.
3x106

Number of antigen sites.

Proximity of red cells.
The London and South East Joint Technical

Slide 16 of 52

Factors Affecting
Antibody/Antigen Reactions.
Ionic strength of the medium.
pH.
Antibody/antigen affinity.
Antigen/antibody concentration.
The London and South East Joint Technical

Slide 17 of 52

Temperature.

The London and South East Joint Technical

Slide 18 of 52

*
Warm and cold agglutinins.
37oC

0-4oC

Cold Antibodies
Warm Antibodies
(mostly IgM).
(mostly IgG).
The London and South East Joint Technical

Slide 19 of 52

Different Antibodies React

Optimally at Different
Temperatures.

ABO antibodies react

optimally at room
temperature or below.

Rh antibodies react
optimally at 370C.
The London and South East Joint Technical

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Incubation Time.

The London and South East Joint Technical

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Antibody/Antigen Reactions
Take Place in Two Distinct
Phases.

Sensitisation.

Agglutination (in vitro).

The London and South East Joint Technical

Slide 22 of 52

Sensitisation, at the
optimal temperature, can
take place in a matter of
microseconds.
Sensitisation at a suboptimal temperature,
however, can take many
hours.
The London and South East Joint Technical

Slide 23 of 52

It is usual, for example, to

incubate tube indirect
antiglobulin tests at 370C
for 15-20 minutes when
the red cells are
suspended in low ionic
strength solution.
The London and South East Joint Technical

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The Number of
Antigen Sites.

3200

3x10

1000

500

47
7000

The London and South East Joint Technical

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*
The number of antigen
sites differs from one
specificity to another,
by the age of the
individual and, even
from individual to
individual.
The London and South East Joint Technical

Slide 26 of 52

The adult A1 red cell has

between 810 000 and 1 170
000 A1 antigen sites per red
cell.

There are only 14 000 copies

of Kidd antigens on each red
cell.
The London and South East Joint Technical

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The adult A1 red cell has

between 810 000 and 1 170
000 antigen sites per red cell.

The red cells of a newborn

will only carry between 250
000 and 370 000 copies of
the A1 antigen.
The London and South East Joint Technical

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The normal D+ red cell will carry 10

000 to 33 000 copies of the D antigen.

An individual with a weak D

phenotype will only carry 200 to 10
000 copies per red cell.

An individual with exulted D (e.g.

D--/D--) will carry between 75 000 and
200 000 copies per red cell.
The London and South East Joint Technical

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Obviously, the
greater the number
of antigen sites, the
more exposed they
are to binding
(sensitisation) by an
antibody.
The London and South East Joint Technical

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Dosage.

Many antigens are expressed on the

red cell more frequently if the genes
are inherited as a double dose
(homozygous).
Examples of such specificities may
react more strongly or readily or only
with, the homozygous red cells.
Examples of anti-M frequently show
dosage.
The London and South East Joint Technical

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Proximity of Red
Cells.

The London and South East Joint Technical

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This Can Be Brought

About in Several
Different Ways.
Centrifugation will increase
gravitational force and bring the
red cells into closer proximity with
each other by mechanical means.
Proteolytic enzymes will bring the
red cells into closer proximity with
each other by chemical means.
Anti-human globulin will bring the
red cells into closer proximity with
each other by immunological
means.
The London and South East Joint Technical

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The London and South East Joint Technical

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The London and South East Joint Technical

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Y = AHG

Y
Y

Y Y

YY

Y
Y

The London and South East Joint Technical

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Presentation of the
Antigen.
Anti-M

Anti-D
12.5nm

Lipid
bilayer
Band 3
Diego

4.2

Ankyrin

GPA
MNS

GPB
MNS

Rh Polypeptide

Rh Glycoprotein
4.1

Spectrin tetramer

Actin

P55

The London and South East Joint Technical

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Ionic Strength of the

Medium.

The London and South East Joint Technical

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Ionic Strength of the

Medium.

The greater the ionic strength

of the medium, the smaller the
zeta potential, and greater the
density of the ionic cloud.
The lower the ionic strength,
the larger the zeta potential,
and the lower the density of
the ionic cloud.
The London and South East Joint Technical

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Ionic Strength of the

Medium.

Lower ionic strengths increase

the likelihood of a successful
approach of a positively charged
immunoglobulin to the antigen
on the red cell surface, as the
density of the positively charged
ionic cloud is reduced. Thus, the
rate of association is increased.
The London and South East Joint Technical

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Ionic Strength of the

Medium.

The rate of association of antibody

with antigen may be enormously
increased by lowering ionic strength.

The initial rate of association

between Anti-D and the D antigen is
increased 1000 fold by reducing the
ionic strength from 0.17 to 0.03.
The London and South East Joint Technical

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Please note,
incidentally, that LISS
stands for low ionic
strength solution and
NOT low ionic
strength saline.
The London and South East Joint Technical

Slide 42 of 52

pH.

The London and South East Joint Technical

*
14

Slide 43 of 52

The equilibrium constant

for most antibodies is
highest between pH 6.5
and 7. Certain antibodies,
however, notably some
examples of anti-M, react
optimally at a greatly
reduced pH
(approximately 4).
The London and South East Joint Technical

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Antibody Affinity.

The London and South East Joint Technical

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Certain antibodies fit

their corresponding
antigens better than
others.
This is to do with the
electric charge on each
antibody and antigen,
together with the degree
of complementarity.
The London and South East Joint Technical

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For example, IgG anti-D

usually fits the D
antigen better than IgG
anti-Jka fits the Jka
antigen.

The London and South East Joint Technical

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Antigen/Antibody
Concentration.

The London and South East Joint Technical

Slide 48 of 52

If the number of antigens

greatly exceeds the number
of antibody molecules
present, there will not be
sufficient antibody molecules
to make an effective lattice
between the antigens on
different red cells, and
agglutination cannot take
place.
The London and South East Joint Technical

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If the number of antibody

molecules present greatly
exceeds the number of
antigen molecules
present, the antibody
molecules will swamp all
the antigen sites, and,
again, agglutination
cannot take place.
The London and South East Joint Technical

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Column Agglutination
Technology (CAT).

CAT has numerous advantages

over conventional tests:
A wash cycle is not required.
Problems with rouleaux are
eliminated.
Results are clear and stable.

The London and South East Joint Technical

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How the Gel Test

Works.

The London and South East Joint Technical

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The London and South East Joint Technical