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:

hazel.chamberssmith@rch.org.au 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?