monoclonal antibodies

1. Monoclonal antibodies production,
applications
Abhishek Sharma
Assistant Professor
L.R Group of Institutes, Solan, Himachal Pradesh

2. Monoclonal antibodies
 An antibody is a protein used by the immune system to identify and
neutralize foreign objects like bacteria and viruses. Each antibody
recognizes a specific antigen unique to its target.
 Monoclonal antibodies (mAb) are antibodies that are identical because
they were produced by one type of immune cell, all clones of a single parent
cell
Characters of Monoclonal Antibodies
 Monoclonal antibodies (mAb) are a 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 a one B-cell lymphocyte
with a myeloma cell.

3. Principle of Hybridoma technology for mAB production
inject the protein into a
mouse.
remove the spleen.
identify cells producing
antibodies.
separate these cells and grow
in tissue culture tubes.
screen each Ab for cross
reactivity.
select the Ab which doesn't
cross react with any other
protein.
 Fusion agent:
Polyethylene glycol
 Medium:
HAT Medium {Hypoxathine-
Aminopterin-Thymidine}

4. Production of mAB by Hybridoma technology
Four basic steps are involved:
1)Immunization
2)Generation of B cells hybridomas by fusing prime b cells and
myeloma cells
3)Selection and screening of resulting clones
4)Cloning by propagating the desired hybridoma

6. Steps: Immunization
• Immunization of animal with immunogen (mixed with an
adjuvant like Al salts, Freund’s complete or incomplete
adjuvant
• The serum of animals is assayed for the relative
concentration, if found to be normal, animal is sacrificed
and spleen which contains the large number of plasma
cells is dissociated into single splenocytes by mechanical
or enzymatic method

7. Steps: Cell fusion
• Splenocytes are mixed with myeloma cells (HGPRT) in an
appropriate medium and is exposed to high concentration of PEG for
short period and fusion is allowed to proceed
• The use of HGPRT cells (that cannot grow in HAT medium) assured
that only hybridomas (hybrid myeloma spleen cells) are selected
• HGPRT, Ab-
myeloma cell was chosen as ideal fusion partner. This
fusion partner has the immortal properties of a cancer cell but does
not secrete its antibody or gene product. Hybridomas generated with
this fusion partner thus secrete only the antibody from the B cell
partner.
• These hybridomas can be propagated in tissue culture to give rise to
large clones secreting homogenous monoclonal antibodies

8. Steps: Selection and screening of
the clones for the monoclonal
antibody specificity
• HAT medium allow only hybridomas cells to grow
• They are removed from the tissue culture flasks, and transferred to
regular culture medium and aliquots are distributed among the well of
96-well plastic culture plates.
• The supernatant of each hybridoma culture can be assayed for a
particular antigen specificity by two methods, i.e., ELISA or RIA

9. Steps: Cloning of hybridomas
secreting specific monoclonal
antibody
• Single cells secreting the desired antibody are isolated from positive
cultures and propagated into cell lines. Two cloning techniques are most
widely employed:
• Limiting dilution (Cells in a culture are enumerated, diluted and aliquoted
into new wells so that only one cell found in any well. Cells are allowed to
regrow and the procedure is repeated several times to increase the
probability that all cells in a given culture are monoclonal)
• Soft agar (many malignant cells may grow, forming spherical colonies, in a
semisolid medium containing low amounts of agar. If the culture can be
reliably dispersed into single cells and the cell concentration is such that
the colonies will be well spaced, then visible colonies picked out of the
agar are likely to be monoclonal

10. Applications of mAB: Diagnostic
1) MAbs in pregnancy testing
Beta HCG ABs
Immunoprecipitation (Micral test): It is
based on the colour shift of monoclonal
antibody to human albumin labelled with
gold

11. 4) mABs IN IMMUNODIAGNOSTIC TESTS
Monoclonal antibodies can also be used
to purify a substance with techniques
called immuno-precipitation and
affinity chromatography.

12. Application of mABs: Therapeutic
1)General
Radioisotope immunoconjugates
mABs conjugates deliver cytotoxic doses of radioactivity to target cells.
Choice of isotopes is critical. Some currently evaluated isotopes are
.Most studies are used β emitters. mABs carrying these emitters are
able to kill nearby Stauder’s cells which cause damage to nearby non-
target cells.
Toxin drug conjugate
mABs are being evaluated for the delivery of potent toxin and drugs to
target cells while reducing toxicity to non- target tissues. Methods
involved is an exclusion of intrinsic infectivity and incorporation of
selectivity for presentation to target cells
2) Transplantation
Organ
Bone marrow
Acute rejection of organ transplants by inhibiting Interleukin-2 on
activated T cells

13. 3) Infectious disease
Micro-organism
mABs against micro-organism can be readily made. mABs can be
used for identification, isolation, detection, structural investigation and
genetic variation and classification of micro-organism.
Parasites
Most important among this class is the malarial parasite. vaccination
against this widespread disease is difficult due to complex life cycle of
these organism. mABs when available will help in treatment,
interception and inactivation pd the parasite life cycle
viruses Bacteria
Influenza Streptococcus A
Rabies Streptococcus B
Hepatitis B Neisseria gonorrhoeae
Herpes Simplex Pseudomonas aeruginosa
Epstein- Barr M.tuberculosis

14. 4) Cardiovascular disease
A murine Mab, 7E3, specific for glycoprotein II b/Iia fibrinogen receptor of
platelets has been evaluated by Centocor
5) Autoimmune disease
mAB directed against B and T lymphocytes are currently being evaluated for
autoimmune diseases.
6) Cancer
1)Radioimmunotherapy (RIT):
Involves the use of radioactively conjugated murine antibodies against
cellular antigens to limit radiation exposure.
Murine antibodies were especially chosen, as their high immunogenicity
promotes rapid clearance from the body.
Ex:Tositumomab in non-Hodgkins lymphoma
2)Antibody-directed enzyme prodrug therapy (ADEPT)
It involves the application of cancer associated monoclonal antibodies which
are linked to a drug-activating enzyme.
Subsequent systemic administration of a non-toxic agent results in its
conversion to a toxic drug, and resulting in a cytotoxic effect which can be
targeted at malignant cells

15. 3)Immunoliposomes: are antibody-conjugated liposomes.
Liposomes - carry drugs or therapeutic nucleotides - conjugated with
monoclonal antibodies - directed against malignant cells.
Tissue-specific gene delivery using immuno-liposomes has also been
achieved in brain, and breast cancer tissue

16. 1) Lymphocyte phenotyping
mABs specific for membrane proteins can be applied to identify various
stages of lymphocyte differentiation. Eg. T helper cell express CD8
membrane protein in both human and mice.
Purification of protein
mABs can be made to any minor protein (P) in a complex mixture since
any Hybridoma clone that secretes antibody to proteins other than P are
eliminated during the screening phase of monoclonal production. This
mABs to a particular protein can be used for its purification
Radioimmunoassay
mABs have the potential to replace or supplement conventional antibodies
in a series of assay. mABs reacts with different sites of single molecules
allows to produce highly specific radio-assays in which one of mABs is
Application of mABs: Analytical