ANTIGEN AND

ANTIBODIES IN BLOOD
BANKING
Marya Saadullah Khan
BLOOD BANKING I
UIMLT
Lecture # 5
 Antigen
Definition:
Antigens are defined as substances recognized by the body as
foreign, causing the body to produce an antibody to react specifically
with it.
Characteristics of antigens:
In order to be an antigen to you it must be foreign (not found in the
host): THE MORE FOREIGN THE BETTER ANTIGEN!
Autologous antigens are your own antigens (not foreign to you)
Homologous, or allogenic, antigens are antigens from someone else
(within the same species) that are foreign to you.
• Antigens must be chemically complex.
• Proteins and polysaccharides are antigenic due to their complexity.
On the other hand, lipids are antigenic only if coupled to protein or
sugar (polysaccharides).
• Besides being chemically complex, antigens must also be large
enough to stimulate antibody production. Their molecular weight
needs to be at least 10,000 kDa.
• Due to the complexity of these molecules there are specific antigenic
determinant sites, or epitopes, which are those portions of the
antigen that reacts specifically with the antibody.
Factors determining whether an antigen
will stimulate an antibody response:
1. Degree of foreignness. Only human blood is transfused to humans.
2. Size and complexity. Although red cells are smaller than white blood cells, they
tend to be more antigenic due to the complexity of the antigens on the cell
surface. Some are proteins and others are oligosaccharides.
3. Dose of antigen administered. How much antigen is the individual exposed to
and what is the frequency of that exposure.
4. Genetic makeup of host may also dictate whether an antibody is produced.
Some individuals have a greater ability to make antibody and others have the
antigen so they would not make the antibody.
 Blood group antigens:
There are over 300 known blood group
antigens
•Over 1,000,000 different antigen sites on each
red blood cell.
•These are attached to proteins or lipids on the
red cell membrane and are usually complex
sugar groups.
•Some stick out far on the red cell membrane
and some are buried within crypts on the
membrane surface.
 Antibodies:
Definition:
Proteins produced by lymphocytes as a result of stimulation by an
antigen which can then interact specifically with that particular antigen.
 Serum protein components
•Human serum can be separated into albumin
and globulin components
•Globulins can be separated into several different
parts:
a. Alpha 1 and alpha 2 globulins
b. Beta globulins (serum complement)
c. Gamma globulins (immunoglobulins or
antibodies)
 Parts of an antibody:
1. Heavy chains - made of alpha, gamma, delta, mu, or epsilon chains
2. Light chains - made of kappa or lambda chains
3. Disulfide bonds - hold chains together
4. Hinge region - allows antibody to flex to reach more antigen sites
5. Fab fragments - contains variable portion of antibody: antigen-
binding sites
6. Fc fragment - contains constant portion of antibody; also site of
complement activation
 Classes of antibodies
1.IgG - provides long-term immunity or protection

2.IgM - first antibody produced in response to an

antigenic stimulus
3.IgA - found in secretions. Protects against
infections in urinary, GI, and respiratory
tracts
4.IgE - involved in allergic reactions
5.IgD - not much known about it. Surface receptor
of B lymphocytes

Most important classes of antibodies in blood

banking are IgM and IgG, and to a certain
extent IgA
Characteristics of IgG and IgM antibodies
1 Clinical significance
• Clinical importance of red cell antibodies in blood bank depend
on whether they can cause in vivo hemolysis, which in turn will
cause transfusion reactions or hemolytic disease of the newborn.

• IgG will frequently cause in vivo[in] hemolysis due to antibody

coating the red blood cells.

• IgM, with a few important exceptions, usually does NOT cause in

vivo hemolysis. The most important of these exceptions are ABO
antibodies.
2. Size of the antibodies
• IgG is relatively small since it is comprised of only one immunoglobulin
subunit. (monomer)
• IgM is relatively large since it is comprised of 5 immunoglobulin subunits.
(pentamer)

• 3. Serum concentration
• IgG is found in the largest concentration of all immunoglobulins in the
plasma.
• IgM is found in relatively small amounts
• IgG > IgA > IgM
4. Complement activation
IgG = will do it if conditions are optimal
IgM = very good complement activator

5. Placental transfer
IgG is small enough to easily cross placenta and is the only
immunoglobulin capable of doing so.
IgM and the other classes do not cross placenta
6. Optimum temperature of reactivity
a. IgG = 37oC
b. IgM = 4 oC (may react at any temperature
below 30C)

7. Number of antigen-binding sites

IgG has 2 binding sites
IgM has 10 binding sites
 Monoclonal antibodies
Monoclonal antibodies react with very specific antigenic
determinants and therefore shows no cross-reactivity. They are
not produced in humans or animals, but harvested from cells in
cells grown in tissue culture.
The tissue culture cells made from fusion of a plasma cell, which
is the antibody producer and the myeloma cell, which provides
longevity and ability to make large amounts of antibody.(known
as Hybridoma)
Monoclonal antibodies used in most reagent antisera today
because they contain high concentrations of highly specific
antibodies and lack infectious disease hazards.
 ANTIGEN-ANTIBODY REACTIONS IN
GENERAL

Rules of Thumb For in vivo Antigen-Antibody Reactions

1. If a person's cell have the antigen, the antibody should
NOT be present in that person's serum
2. If an antibody to a blood group antigen is present in
the serum of a person, his or her cells should lack that
antigen
3. The antigens are on the cells and the antibodies are in
the serum
 Stages of Antigen-Antibody Interaction
1. The first stage is sensitization. Sensitization occurs when antibodies
react with antigens on the cells and coat the cells.
2. The second stage of the reaction is agglutination. Agglutination occurs
when antibodies on coated cells form cross-linkages between cells
resulting in visible clumping.
 FACTORS AFFECTING
SENSITIZATION (In vivo or in vitro)

1.Specificity depends on the spatial and chemical "fit" between antigen and
antibody
2. Since the immunoglobulins and the red cell membranes both have an electrical
charge, there is an optimum pH. pH differences cause differences in chemical
structures of antigens/antibodies, affecting the "fit".
3. The optimum temperature depends on the type of antibody involved. IgG
antibodies react best at 37oC; IgM react best at 4oC.
 4. Optimum incubation time: you need to incubate long enough to
reach equilibrium, but not too long

5. The antigen's accessibility is also

important since the antibodies must be
able to reach antigens. Those antigens,
like the ABO antigens, are on the
surface of the red cell while others may
be hidden in the crypts of the cell
membrane.
FACTORS AFFECTING AGGLUTINATION IN VITRO

1. Number of Antigen Sites

The number of antigen sites on the red cell is important since the
more antigen sites result in more antibodies being attached and
forming cross-linkages. These cross-linkages result in agglutination
2. Size and Structure of the Antibody
The larger antibodies (IgM) can reach between more antigen sites
on different red 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.
3. Distance between Cells
Centrifugation of the cells attempts to bring the red blood cells closer together,
but even then the smaller IgG antibodies usually can not reach between two
cells. The larger antibodies, IgM, can reach between cells that are further apart
and cause agglutination.
 Antigen-Antibody Ratio
The optimum ratio is 80 parts antibody to 1 part antigen. There are
specific terms for variations in this ratio.
Prozone - antibody excess:
Antibodies saturating all antigen sites; no antibodies forming cross-
linkages between cells; no agglutination
Zone of equivalence:
antibodies and antigens present in optimum ratio, agglutination formed
Zone of antigen excess (Post-zone):
too many antigens - any agglutination is hidden by masses of
unagglutinated antigens
In order to get optimum
antigen-antiboy
concentration in Blood
Banking we make
washed 3% saline
suspension of red cells
to mix with our
reagents.
 Terms used to describe antibodies
Immunoglobulin:
antibody formed as a result of immune stimulus (exposure to foreign
antigen)
Naturally occurring
antibody formed without prior exposure to foreign antigen
Autoantibody:
antibody formed to one's own antigens (abnormal condition)
Alloantibody (unexpected, irregular, atypical):
antibody formed to foreign antigens, but within the same species
Agglutinin:
antibody capable of causing agglutination when reacting with
corresponding antigen
 Terms used to describe
Isoagglutinin:
antibodies
name commonly given to blood group antibodies anti-A and anti-B

Saline agglutinin:
antibody capable of causing direct agglutination of antigens suspended in a
saline medium without requiring any enhancement techniques

Hemolysin:
antibody capable of causing hemolysis when reacting with corresponding
antigen

Cold antibody (cold agglutinin):

antibody whose optimal temperature of reactivity is less than 30 oC

Warm antibody:
antibody whose optimal temperature of reactivity is greater than 35 oC