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TRANSCRIPT
Antibodies – Polyclonal Vs Monoclonal
Affinity, cross-reactivity, reaction rates, stability
and handling of antibodies
Hazel Chambers-Smith,
Sri Lanka Workshop of Basics of Immunohistochemistry, June 2019
Before we start
• If I speak too fast please let me know
• Please feel free to ask questions at any
time
• If I don’t know the answer please email
me at:
[email protected] and I
will find out and get back to you
The team in Melbourne
• Let us begin
What are Antibodies?
• Antibodies are produced in response to
a stimulus.
• The stimulus can be bacteria, fungi,
viruses and foreign cells
• These stimuli are called antigens
• Antibodies bind to antigens and are not
easily dislodged once bound
• The specificity and high binding strength
make them popular for reagents in
diagnosis
• Animals produce
antibodies
• Produced by plasma
cells or transformed B
Lymphocytes
Antibody structure
• Consist of two basic
units:
• A pair of light chains
(kappa or lambda)
• A pair of heavy chains
(gamma, alpha, mu,
delta or epsilom)
What makes up an antigen?
• Antigens are made up of
a lot of individual sites
• Each individual site of the
molecule is called an
antigenic determinant OR
epitope
• Note that the rigid parts of
an antibody can also
serve as an antigen… We
will investigate this later!
Polyclonal antibodies
• Produced by immunizing an animal with
a purified molecule (immunogen)
• Immune response can be harvested by
bleeding the animal to obtain the serum
(rich in immunoglobulins)
• Many different clones of plasma cells
will be produced in response to the
many epitopes on the immunogen.
• Also important to bleed the animal prior
to sensitization with immunogen to get a
baseline for negatives
• Be aware that the polyclonal antibodies
may also include a range of antibodies
to a whole range of antigens the animal
was exposed to before the sensitization
• This can lead to high non-specific
binding in a test environment
Monoclonal antibodies
• Developed by hybridoma technique by
Kihler and Milstein in 1975
• Combines plasma cells or transformed B
cells with an immortal, neoplastic
myeloma cell line
• With cloning, the cell line can be cultured
or in ascites “forever”.
• The purity of the cell line can be enhanced
by removing hybrids reacting to unwanted
antigens/epitopes during screening
Monoclonal V Polyclonal
• Monoclonal are more specific and only
react to one part of the antigen
• Monoclonal are less likely to cross-react
with other proteins
• Monoclonal are not contaminated with
any other antibodies
• Monoclonal will always be identical
whereas Polyclonal will vary
Can Monoclonals be too specific?
• As Monoclonals are raised to react with
a specific epitope, this can sometime be
too precise
• In autoimmune reactions, the exact
immunoglobin binding may not be
known so rather than test with 3-4
different monoclonal antibodies, (IgG,
IgM, IgA etc), a polyclonal able to bind
with all Ig subclassed can be used as
one test.
Affinity
• Defined as the three-dimensional fit of
the antibody to its specific antigen and
the strength of the binding between the
epitope and its specific antibody-
combining site
• Antigen-antibody binding is reversible
• High affinity antibodies should be
chosen
• Factors that weaken the antigen-
antibody bond such as
• High salt concentrations,
• high temperature and
• very low pH
During the washing of the specimens
should be avoided.
Cross-reactivity
• The term “cross-reactivity” denotes an
immunochemical activity that can occur
either
• between an antibody and two or more
antigens or
• vice versa, when an antigen reacts with
several different antibodies.
• Can also be induced through antigen
retrieval methods leading to loss of
specificity to the antigen
PAX8 and PAX5 cross-
reactivity
Reaction Rates
• The binding of an antibody to its antigen
is reversible
• Rate of binding is proportional to the
rate of the reactants
• Rate of reaction can be sped up with
the use of high concentrations of high
affinity antibodies and detection
systems
• At equilibrium, the rate of
antibody:antigen complex formation is
equal to the rate of dissociation into its
components of antibody and antigens
• This can be measured!
• It is known as an affinity constant (1/KD)
• The smaller a KD value, the higher the
affinity the antibody has for the target
• A high affinity antibody: 10-9 to 10-12
• Most are 10-6 to 10-9
Stability and storage of antibodies
• “Shelf-Life” is determined by
manufacturers by testing their product
over a period of time.
• A manufacturer does not need to test
the product until it loses affinity, but just
needs to state the period of time that the
product has been tested.
• An antibody may be tested by the
manufacturer for 18 months, but the
useful life may be 10+ years!
Polyclonal stability
• Less stable as an immunoglobulin
fraction than as whole serum
• Less exposure during manufacture to
extremes in pH, high or low salt
concentrations shows better antibody
stability
Monoclonal stability
• Method of purification is important in
determining stability
• IgG and IgM subclasses of antibodies
are especially sensitive to changes in
stability
Storage of antibodies
• Some can be aliquoted and frozen (refer
to data sheets!)
• Laboratory must confirm that the
antibody is active and appropriate for
use on patient tissue
• Keep logs of invoices, date of receipt,
batch numbers and expiry dates
• Always store the antibodies as per the
manufacturers instructions.
Expiry dates
• Some antibodies don’t come with an
expiry date.
• The rule for these is 12 months from
date of receipt
• Have a system in place to log this
Maintaining Ab quality
• Avoid repeated freeze/thaw cycles
• Avoid excessive heat
• Bring frozen solutions to temperature
slowly
• Return reagents to proper storage
conditions promptly
Unstable antibodies
• Some are so fragile that it is warranted
to dilute the antibody fresh for each run
• Should only be accommodated when
there is no other choice
• It seems that perhaps the 4 degree
storage or the diluent and antibody may
be incompatible
Handling of antibodies
• Concentrates come in small vials
• From 0.1ml (100ul) to 1ml (1000ul)
• Take caution and only have one
antibody out at a time
• The vials are easy to knock over
• Always dispense diluent first and then
concentrate into diluent
• Concentration is critical-unwanted
cocktails are easily achieved!!
Any Questions?