production and applications of monoclonal antibodies, monoclonal antibodies ,applications of monoclonal antibodies, production of monoclonal antibodies,

1. Production and
applications of
monoclonal
antibodies
PRESENTED BY
S.KAAYATHRI DEVI
19PO03
II M.SC., BIOTECHNOLOGY

2. Antibodies
 Antibodies are large Y-shaped proteins.
 They are recruited by the immune system to
identify and neutralize foreign objects like
bacteria and viruses.
 Each antibody has a unique target known as
the antigen present on the invading organism.
 This antigen is like a key that helps the
antibody in identifying the organism.
 The specific region on an antigen that an
antibody recognizes and binds to is called the
epitope, or antigenic determinant.

3. Monoclonal antibodies
 Monoclonal antibodies are identical immunoglobulins, generated from a
single B-cell clone. These antibodies recognize unique epitopes, or binding
sites, on a single antigen.
 Derivation from a single B-cell clones and subsequent targeting
of a single epitope is what differentiates monoclonal antibodies
from polyclonal antibodies.
 Polyclonal antibodies are antibodies that are derived from different cell
lines. They differ in amino acid sequences.

4. Characters of monoclonal
Antibodies
 Monoclonal antibodies (mAB) are single type of antibody that are identical
and are directed against a specific epitope (antigen, antigenic determinant)
and are produced by B-cell clones of a single parent or a single hybridoma cell
line.
 A hybridoma cell line is formed by the fusion of one B-cell lymphocyte with a
myeloma cell.
 Some myeloma cell synthesize single mAB antibodies naturally.

5. Advantages of using
Monoclonal Antibodies:
 Though expensive, monoclonal antibodies are cheaper to develop than
conventional drugs because it is based on tested technology.
 Side effects can be treated and reduced by using mice-human
hybrid cells or by using fractions of antibodies.
 They bind to specific diseased or damaged cells needing treatment.
 They treat a wide range of conditions.

6. Disadvantages of using
Monoclonal Antibodies:
 Time consuming project - anwhere between 6 -9 months.
 Very expensive and needs considerable effort to produce them.
 Small peptide and fragment antigens may not be good antigens
 Monoclonal antibody may not recognize the original antigen.
 Hybridoma culture may be subject to contamination.
 System is only well developed for limited animal and not for other
 animals.
 More than 99% of the cells do not survive during the fusion process
reducing the range of useful antibodies that can be produced against an
antigen
 It is possibility of generating immunogenicity.

8. Step – 1: Immunization of the
mice
 Mice are immunized every 2-3 weeks
with an antigen that is prepared for
injection

9. Step 2: Screening of
Mice for Antibody
Production
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.

10. Step – 3 : Isolation of antibody
producing spleen cells
 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.

11. Step 4 : Production of
hybridomas
 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.

12. Step 5 : Screening of
hybridomas

14. Step 6 : Culturing
hybridoma cells
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. Step 7 : Screening for
desired antibodies
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. Step 8 : Selection and
culture of screened
antibodies
Finally, the desired antibodies are grown in mass culture and
are frozen for storage.

20. Biochemical analysis
 Routinely used in radioimmunoassay (RIA) and enzyme-linked
immuno sorbent assays (ELISA) in the laboratory.
 These assays measure the circulating concentrations of hormones
(insulin, human chorionic gonadotropin, growth hormone, progesterone,
thyroxine,triiodothyronine, thyroid stimulating hormone) and several other
tissue and cell products (blood group antigens, blood clotting
factors, interferon’s, interleukins,tumormarkers).
 Eg. Pregnancy by detecting the urinary levels of human chorionic gonadotropin.
 Hormonal disorders analysis of thyroxine, triiodothyronine.
 Cancers estimation of plasma carcinoembryonic antigen in colorectal cancer,
and prostate specific antigen for prostate cancer

21. Diagnostic imaging
 Radiolabeled—MAbs are used in the diagnostic imaging of diseases,
and this technique is referred to as immunoscintigraphy. The radioisotopes
commonly used for labeling MAb are iodine—131 and technetium—99. The
MAb tagged with radioisotope are injected intravenously into the patients.
 These MAbs localize at specific sites (say a tumor) which can be detected by
imaging the radioactivity. In recent years, single photon emission computed
tomography (SPECT) cameras are used to give a more sensitive three
dimensional appearance of the spots localized by radiolabeled— MAbs.
 Myocardial infarction, DVT, atherosclorosis etc.

28. Reference
 https://www.slideshare.net/DrxSurajMandal/monoclonal-
antibodies-preparation-application
 https://www.slideshare.net/sakshisaxena9256/production-
of-monoclonal-antibodies-and-its-application-rituximab
 https://www.slideshare.net/santupolley/monoclonal-
antibody-33554401
 Pictures from Google Inc.,(all the images used in this
presentation are taken from Google search and slideshare)