Antibodies are proteins produced by plasma cells that bind to specific antigens. Monoclonal antibodies bind to a single epitope of an antigen, whereas polyclonal antibodies bind to multiple epitopes. Monoclonal antibodies are produced through cell fusion, where antibody-producing spleen cells are fused with myeloma cells to form immortal hybridoma cell lines that each secrete a single antibody clone. This process allows continuous production of homogeneous antibodies against a specific epitope. In contrast, polyclonal antibodies come from antiserum containing a heterogeneous mixture of antibodies against multiple epitopes on an antigen.

2.  Antibodies are globular proteins or Immunoglobulins
synthesized by a type of white blood cell called plasma cells
against the antigen that stimulated its production.
 The region of antibody that interact with antigen is
called Paratope.
 Antigen antibody interaction is specific.
ANTIBODY

4. A monoclonal antibody refers to an antibody
normally recognizing only a single antigen (e.g. a single
protein) and within which only a single common epitope
is recognized.

5. Figure: Polyclonal and Monoclonal Antibodies

6. The idea of ‘magic bullet’
was first proposed by Paul
Ehrlich who at beginning of
20th century postulated that “if a
compound could be made that
selective targeted a disease
causing organism than a toxin
for that organism could be
delivered along with agent of
selectivity”.

8. Figure : Monoclonal antibodies (mAbs) production

9.  Mice are immunized every 2-3 weeks with an antigen
that is prepared for injection

10. Blood samples are
obtained from mice for
measurement of serum
antibodies whose titer is
determined with various
techniques,
such as enzyme-linked
immuno sorbent assay
(ELISA) and flow
cytometry.

11.  When the antibody titer is high enough, mice are
commonly boosted by injecting antigen without
adjuvant intra peritoneally or intravenously (via the
tail veins) 3 days before fusion but 2 weeks after the
previous immunization.
 If the titer is too low, mice can be boosted until an
adequate response is achieved, as determined by
repeated blood sampling.
 Then the mice are euthanized and their spleens
removed for in vitro hybridoma cell production.

12.  A week before cell fusion, myeloma cells are grown in 8-
azaguanine (analog of guanine, acts as competitive
inhibitor).
 Myeloma cells lack HPGRT (hypoxanthine
phospho ribosyl transferase) enzyme, which is
responsible for synthesis of nucleotides.
 The cells are then screened in HAT (hypoxanthine-
aminopterin- thymidine) medium which blocks the
pathway for nucleotide synthesis.

13. Step 5 : Screening of hybridomas

14.  Hybridomas are separated and individually cultured : 1 cell per
well
 These cells are called as clonal culture.
 Because each cell in the well is derived from singe cell and are
therefore identical.
 After few weeks, when growing cultures can be seen, further
screening can be done for desired antibody.

15.  Antigens are immobilized in the
wells and the antibodies are
transferred (one per well) so
that they bind to the
complementary antigen.
 Different antibodies react to
different epitopes on the same
antigen.

16. Finally, the desired antibodies are grown in mass culture
and are frozen for storage.

17. A polyclonal antibody refers to an
antibody normally recognizing only a
single antigen but within which a
number of different epitopes are
recognized.

18.  Antibodies used for research and diagnostic purposes are
often obtained by injecting a lab animal such as a rabbit
with a specific antigen.
 Within a few weeks, the animal’s immune system will
produce high levels of antibodies specific for the
antigen.
 These antibodies can be harvested in an antiserum,
which is whole serum collected from an animal
following exposure to an antigen.

19.  Because most antigens are complex structures with multiple
epitopes, they result in the production of multiple antibodies in
the lab animal.
 This so-called polyclonal antibody response is also typical of the
response to infection by the human immune system.
 Antiserum drawn from an animal will thus contain antibodies from
multiple clones of B cells, with each B cell responding to a specific
epitope on the antigen.

20.  Antiserum obtained from animals will not only contain
antibodies against the antigen artificially introduced in the
laboratory, but it will also contain antibodies to any other
antigens to which the animal has been exposed during its
lifetime.
 For this reason, antisera must first be “purified” to remove
other antibodies before using the antibodies for research or
diagnostic assays.

21. Fi
Figure: The process for harvesting polyclonal antibodies
produced in response to an antigen.

22. Monoclonal antibodies Polyclonal antibodies
Expensive production Inexpensive production
Long production time Rapid production
More Specificity less Specificity
Recognize a single epitope on an
antigen
Recognize multiple epitopes on an
antigen
Production is continuous and uniform
once the hybridoma is made
Different batches vary in composition
Few host species options Many host species options
A homogenous antibody population. A heterogeneous antibody population.

23. Thank you