BIOTECHNOLOGY IS CHALLENGING SUBJECT TO TEACH AND UNDERSTAND ...... ITS A VERY INTERESTING TO LEARN ABOUT HYBRIDOMA TECHNOLOGY .. THEIR PRODUCTION AND APPLICATION ALSO ....

1. MONOCLONAL
ANTIBODIES
By
TEJASWINI L. ASAWE
ASSISTANT PROFESSOR
SIDHHIS INSTITUTE OF PHARMACY
THANE

2. Monoclonal antibodies
 History of Hybridoma Technology.
 Hybridoma technology is a well-established
method to produce monoclonal antibodies
(mAbs) specific to antigens.
 Hybridoma cell lines are formed via fusion
between a short-lived antibody-producing B
cell and an immortal or living myeloma cell.
 Myeloma is develops from cells in the bone
marrow blood cancer that called plasma cells.

3. HISTORY
 Hybridoma technology was discovered in 1975 by
two scientists, Georges Kohler and Cesar Milstein
 They wanted to create immortal hybrid cells by
fusing normal B cells from immunized mice with
their myeloma cells.
 By cloning individual hybrid cells, they established
the first hybridoma cell lines which can produce
single type of antibody specific to the specific
antigen.
 Their discovery is considered one of the greatest
breakthroughs in the field of biotechnology.

4. Advantages
 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 cell
fragments or small amount of antibodies.
 They bind to specific diseased or damaged
cells needing treatment.
 They treat a wide range of conditions

5. Disadvantage
 It is Time consuming process takes time 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.

6. Steps Involved in Hybridoma
Technology
 Hybridoma technology is composed of several
technical procedures, including
 antigen preparation,
 animal immunization,
 cell fusion,
 hybridoma screening and
 sub cloning,
 characterization and
 production of specific antibodies

7. (1) Immunization of a mouse
(2) Isolation of B cells from the
spleen
(3) Cultivation of myeloma cells
(4) Fusion of myeloma and B cells
(5) Separation of cell lines
(6) Screening of suitable cell lines
(7) in vitro (a) or in
vivo (b) multiplication
(8) Harvesting

8. 1) Cell fusion
 Laboratory animals (mammals, e.g. mice) are first exposed to the antigen
against which an antibody is to be generated.
 These injections are typically followed by the use of in
vivo electroporation,
(electroporation process is done by using a electric pulse to transfect the
cells with DNA )which significantly enhances the immune response.
 Once splenocytes are isolated from the mammal's spleen, the B cells are
fused with immortalised myeloma cells.
 The fusion of the B cells with myeloma cells can be done using
electrofusion.
 Electrofusion causes the B cells and myeloma cells to align and fuse
with the application of an electric field.
 Alternatively, the B-cells and myelomas can be made to fuse by chemical
protocols, most often using polyethylene glycol.
 The myeloma cells are selected beforehand to ensure they are not

9. 2) Hybridoma screening
 Fused cells are incubated in HAT medium (hypoxanthine-
aminopterin-thymidine medium) for roughly 10 to 14 days.
 Aminopterin blocks the pathway that allows for nucleotide synthesis.
 Hence, unfused myeloma cells die, as they cannot produce
nucleotides because they lack HGPRT.
 Removal of the unfused myeloma cells is necessary because they
have the potential to outgrow other cells, especially weakly
established hybridomas.
 Unfused B cells die as they have a short life span.
 In this way, only the B cell-myeloma hybrids survive, since the
HGPRT gene coming from the B cells is functional.
 These cells produce antibodies (a property of B cells) and are
immortal (a property of myeloma cells).
 The incubated medium is then diluted into multi-well plates to such an
extent that each well contains only one cell.
 Since the antibodies in a well are produced by the same B cell, they
will be directed towards the same epitope, and are thus monoclonal
antibodies.

10. 3) MONOCLONAL ANTIBODY
PRODUCTION
 The next stage is a rapid primary screening process, which
identifies and selects only those hybridomas that produce
antibodies of appropriate specificity.
 The first screening technique used is called ELISA.
 The hybridoma culture supernatant, secondary enzyme labeled
conjugate, and chromogenic substrate, are then incubated, and
the formation of a colored product indicates a positive hybridoma.
 Flow cytometry (flow of particles study ) screening has been the
local form of the antigen on the cell surface.
 Flow cytometry is used to detect and analyze the chemical and
physical characteristics of cells or particles.
 In the flow cytometry-based screening, a mixture of antigen-
negative cells and antigen-positive cells is used as the antigen to
be tested for each hybridoma supernatant sample.

11.  The B cell that produces the desired antibodies can be cloned to
produce many identical daughter clones.
 Once a hybridoma colony is established, it will continually grow in
culture medium and produce antibodies.
 Multiwell plates are used initially to grow the hybridomas, and
after selection, are changed to larger tissue culture flasks.
 This maintains the well-being of the hybridomas and provides
enough cells for cryopreservation (Biological material preserve
at −80 °C (−112 °F) or −196 °C (−321 °F) using liquid nitrogen) .
 the supernatant is the liquid found above a precipitate or
sediment.
 The culture supernatant can yield 1 to 60 μg/ml of monoclonal
antibody, which is maintained at -20 °C or lower until required.
 By using culture supernatant or a purified immunoglobulin
preparation, further analysis of a potential monoclonal antibody
producIng hybridoma can be made in terms of reactivity,
specificity, and cross-reactivity

12. HAT - medium (hypoxanthine-aminopterin-thymidine medium
hypoxanthine-guanine phospho ribosyl transferase (HGPRT) gene,

13. Application
 Pregnancy Testing
 MAbs that have been developed to detect
human chorionic gonadotropin (HCG) are now
present in pregnancy test kits.
 Radioimmuno detection (RID) of Cancer
 An imaging technique used to detect the
presence of cancerous or cancer-specific cells
has been produced as mAbs.

14.  Treatment of Cancer through Drugs
 Many different drugs are being developed in
clinical trials used to treat various strains of
cancer.
 In fact, some of these are already on the
market.
 In 1997, a drug named Ritoxin was approved
by the FDA for commercial use which is based
on mAb technology.

15. Viral Disease Treatment
 Doctors hope that with further research into
mAbs and an increased knowledge of their
properties, treatments will become available for
diseases previously thought to be incurable,
such as AIDS.
 Identifying Pathogens
 MAbs can now be used to identify strains of a
single pathogen, for example neisseria
gonorrhoeae.

16.  Rhesus disease Immunization
 Rhesus disease is condition where antibodies
in a pregnant woman's blood destroy her
baby's blood cells.
 Anti-rhesus antiserum has been made to cure
rhesus disease.

17. References
 1. Zaroff, S., & Tan, G. (2019). Hybridoma technology:
the preferred method for monoclonal antibody
generation for in vivo applications.
 2. Zhang, C. (2012). Hybridoma technology for the
generation of monoclonal antibodies. In Antibody
methods and protocols (pp. 117-135). Humana Press,
Totowa, NJ.
 3. Liu, J. K. (2014). The history of monoclonal
antibody development–progress, remaining
challenges and future innovations. Annals of Medicine
and Surgery, 3(4), 113-116.
 4. Greenfield, E. A. (2018). Polyethylene Glycol
Fusion for Hybridoma Production. Cold Spring Harbor
Protocols, 2018(3), pdb-prot10317