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.
- Besides being chemically complex, antigens must also be
large enough to stimulate antibody production. Their molecular weight
needs to be at least 10,000.
- 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
Factors determining whether an antigen will stimulate an antibody response:
- Degree of foreignness. Only human blood is
transfused to humans.
- 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.
- Dose of antigen administered. How much antigen is the
individual exposed to and what is the frequency of that exposure.
- 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:
are over 300 known blood group antigens
- Over 1,000,000 different antigen sites on each red
- These antigens 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.
Proteins produced by lymphocytes as a result of
stimulation by an antigen which can then interact specifically with that
- 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:
- Heavy chains - made of alpha, gamma, delta, mu, or epsilon chains
- Light chains - made of kappa or lambda chains
- Disulfide bonds - hold chains together
- Hinge region - allows antibody to flex to reach more
- Fab fragments - contains variable portion of antibody: antigen-binding
- Fc fragment - contains constant portion of antibody; also site of
Classes of antibodies:
- IgG - provides long-term immunity or protection
- IgM - first antibody produced in response to an antigenic stimulus
- IgA - found in secretions. Protects against infections in urinary, GI, and
- IgE - involved in allergic reactions
- IgD - not much known about it. Surface receptor of B lymphocytes
- Most important classes of antibodies in blood banking are IgM and
to a certain extent IgA
Characteristics of IgG and IgM antibodies
- Clinical 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 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.
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)
- IgG is found in the largest concentration of all immunoglobulins
in the plasma.
- IgM is found in relatively small amounts
- IgG > IgA > IgM
- IgG = will do it if conditions are optimal
- IgM = very good complement activator
- 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
Optimum temperature of reactivity
- a. IgG = 37oC
- b. IgM = 4 oC (may react at any temperature below 30C)
- Number of antigen-binding sites
- IgG has 2 binding sites
- IgM has 10 binding sites
Terms used to describe antibodies
- antibody formed as a result of immune stimulus (exposure
to foreign antigen)
- Naturally occurring
- antibody formed without prior exposure to foreign
- antibody formed to one's own antigens (abnormal
- Alloantibody (unexpected, irregular, atypical):
- antibody formed to foreign antigens, but within the
- antibody capable of causing agglutination when reacting
with corresponding antigen
- name commonly given to blood group antibodies anti-A and
- Saline agglutinin:
- antibody capable of causing direct agglutination of
antigens suspended in a saline medium without requiring any enhancement
- antibody capable of causing hemolysis when reacting with
- Cold antibody (cold
- antibody whose optimal temperature of reactivity is
less than 30oC
- Warm antibody:
- antibody whose optimal temperature of reactivity is
greater than 35oC
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
Monoclonal antibodies used in most reagent antisera today
because contain high concentrations of highly specific antibodies and lack
infectious disease hazards associated with human-source antiserum.
ANTIGEN-ANTIBODY REACTIONS IN GENERAL
Rules of Thumb For in vivo Antigen-Antibody Reactions
- If a person's cell have the antigen, the antibody
should NOT be present in that person's serum
- If an antibody to a blood group antigen is present in the serum of a
person, his or her cells should lack that antigen
- The antigens are on the cells and the antibodies are in the serum
Stages of Antigen-Antibody Interaction
The first stage is sensitization. Sensitization
occurs when antibodies react with antigens on the cells and coat the
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)
- Specificity depends on the spatial and chemical "fit" between antigen and
- 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".
- The optimum temperature depends on the type
of antibody involved. IgG antibodies react best at 37oC; IgM react best at
- Optimum incubation time: you need to incubate long enough to reach
equilibrium, but not too long
- 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
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
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.
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.
- The concept Zeta potential is important to understand
why the cells will maintain a certain distance from each other. Zeta
potential refers to the repulsion between the red blood cells.It is due to an
electric charge surrounding cells 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
- To overcome zeta potential techniques need to
neutralize these charges One of the common techniques is:
1) Add albumin to test
2) OH- groups of albumin neutralize positive charge
The optimum ratio is 80 parts antibody to 1 part antigen.
There are specific terms for variations in this ratio.
- Prozone -
- Antibodies saturating all antigen sites; no antibodies
forming cross-linkages between cells; no agglutination
- Zone of
- antibodies and antigens present in optimum ratio,
- Zone of antigen excess
- 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.
OBJECTIVES - ANTIGENS AND ANTIBODIES IN BLOOD BANKING
- Define the following terms:
Homologous (allogeneic) antigen
- Describe the characteristics common to all antigens
- List the factors that determine whether an antibody
will be formed in response to an antigen stimulus
- Describe the characteristics of blood group antigens
- Identify the parts of an antibody, and state the
purpose of each part
- Differentiate between:
an autoantibody and an alloantibody
and naturally-occurring antibody
an agglutinin and a hemolysin
a warm and
a cold antibody
- Explain what is meant by "clinically significant"
- List the classes of immunoglobulins
- State the differences between IgG and IgM antibodies
Number of binding sites
Ability to cross placenta
Ability to fix complement
Ability to cause direct agglutination
Optimum temperature of reaction
- Briefly describe how monoclonal antibodies are made
- State the main characteristics of monoclonal antibodies
- Differentiate between sensitization and agglutination
- List five factors that affect sensitization
- List five factors that affect agglutination
- Define specificity in antigen-antibody reactions, and
explain how pH may affect it
- Explain how length of incubation affects antigen-antibody reactions
- Explain how accessibility affects antigen-antibody reactions
- Explain how the number of antigen sites on the red
cells affect agglutination
- Explain how the size and structure of antibodies
affects agglutination of red cells
- Explain what the zeta potential is, and how this may be
overcome to promote agglutination
- Differentiate between prozone, zone of equivalence, and zone of antigen
- State the optimum ratio of antigen to antibody to