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 Antibodies
 An antibody (Ab), also known as an immunoglobulin
(Ig), is a large Y-shaped protein produced by B cells
that is used by the immune system to identify and
neutralize foreign objects such as bacteria and viruses.
 “An antibody produced by a single clone of cells.
Monoclonal antibody is therefore a single pure
type of antibody”
 Each monoclonal antibody recognizes a specific
antigen.
ANTIBODIES
POLYCLONAL MONOCLONAL
 Derived from different B
Lymphocytes cell lines.
 Batch to Batch variation
affecting Ab reactivity &
treatment.
 NOT Powerful tools for
clinical diagnostic tests.
 Derived from a single B cell
clone.
 No Batch to Batch
variations. Effectiveness of
Ab is much more
predictable.
 Enable the development of
secure immunoassay
systems.
 The types of mAb designed
 Murine source mAbs: rodent mAbs with excellent affinities
and specificities generated using conventional hydrioma
technology. Clinical efficacy compromised by HAMA(human
anti murine antibody) response, which lead to allergic or
immune complex hypersensitivities.
 Chimeric mAbs: chimers combine the human constant
regions with the intact rodent variable regions. Affinity and
specificity unchanged. Also cause human antichimeric
antibody response (30% murine resource)
 Humanized mAbs: contained only the CDRs of the rodent
variable region grafted onto human variable region
framework

1) Homogeneity:
Monoclonal antibody represents a single antibody molecule that
binds to antigens with the same affinity and promote the same
effectors functions.
2) Specificity:
The product of a single hybridoma reacts with the same epitope on
antigens.
3) Immunizing Antigen:
Need not be pure or characterized and is ultimately not needed to
produce large quantities of antibody.
4) Selection:
It is possible to select for specific epitope specificities and generate
antibodies against a wider range of antigenic determinants.
5) Antibody Production:
Unlimited quantities of a single well-defined monospecific reagent.
1) Affinity:
Average affinity of monoclonal antibodies are generally
lower than polyclonal antibodies.
2) Effector Functions:
Because antibody is monoclonal, it may not produce the
desired biologic response.
3) Specificity:
Monoclonals against conformational epitopes on native
proteins may lose reactivity with antigens.
4) Cross reactions:
Antibodies sometimes display unexpected crossreactions
with unrelated antigens.
5) Time and effort commitment:VERY LARGE.
 Process by which bulk quantities of targeted antibodies
against a specific antigen are produced.
 Monoclonal antibodies are produced via multiple/identical
copies of a certain cell called a hybridoma.
 To create Hybridoma cells the fusion of 2 cells are needed in
order to combine the characteristics of the 2 cells into 1 cell.
 1 of the cells is a producing cell antibody which is a B-
Lymphocyte used from a laboratory mouse and the other is a
tumor cell named myeloma.
 Tumor cells have the ability to grow indefinitely and at an
exceeding rate from normal cell growth.
 Laboratory produced Hybridoma cells replicate much faster
than normal antibody producing cells, and the individual
hybridomas produce the specific antibodies for an indefinite
period of time.
 Hybridoma cells manufacture the specific monoclonal
antibody that was originally produced by the B-
Lymphocyte cell.
 The original B-Lymphocyte cell will produce the
Monoclonal antibody depending on the kind of antigen
that was injected into the mouse just prior to the
harvesting of the B-Lymphocyte cells.
 Eg: If the mice were injected with a certain virus, the
mouse will have B-Lymphocytes that produce those
specific virual antibodies. Fusion with a tumor cell to
make the hybridoma, result in the production of
monoclonal antibodies against the specific virus.
 The hybridoma cells are placed into media that can help
them grow and produce the bulk quantities of monoclonal
antibodies.
 There are 2 ways for growing monoclonal antibodies, 1 is
to grow them in laboratory flasks meaning In Vitro, and
the other is to grow them in the stomach lining of mice.
 Injecting the hybridomas into the mice is the familiar
method of harvesting monoclonal antibodies. This
method is done by mixing spleen cells from the mouse
that has been immunized with the desired antigen with
myeloma cells. The myeloma cells need to have lost their
ability to synthesize HGPRT enzyme (hypoxanthine-
guanine-phosphoribosyltransferase) which enables the
cells to synthesize purines using an extracellular source of
hypoxanthine as a precursor.
 Cells have another pathway that they synthesize
purines so lack of HGPRT is not a problem for the cell
but when cells are exposed to aminopterin they are
unable to use this other pathway and are fully
dependent on HGPRT for their survival. So to
summarize, unfused myeloma cells can’t grow since
they lack HGPRT and unfused normal spleen cells
can’t grow since they have a limited life-span.
Hybridoma cells are grow indefinitely since the spleen-
cell copartner supplies HGPRT and the myeloma
partner is immortal.
 The first step is transferring of the cell fusion mixture to HAT culture
medium which contains hypoxanthine, aminopterin & pyrimidine
thymidine.
 The 2nd step is testing the supernatants from each culture in order to
locate the producing the desired antibody.
 One must isolate the single cells from each antibody-positive culture
and subculture them, this represents the clone which its antibodies
are monoclonal.
 Every single cell culture secretes a specific kind of antibody that is
directed against a certain determinant/selected antigen.
 The 3rd step is scaling up the size of the cultures of the successful
clones.
 Hybridoma cultures can be grown indefinitely in vitro in culture
vessels which yield 15-65 µg/ml and in vivo using mouse, where the
antibody concentration in the serum 0.5-15 mg/ml.
 In the past years, animal welfare activists in worldwide are trying to
limit the use of mice for the production of monoclonal antibodies.
 When the monoclonal antibody is produced it can be used as a
probe to track down, bind to and purify the specific protein that
induced its formation.
1) FILTRATION METHOD:
Cells, cell debris,
lipids, and clotted
material are first
removed, typically by
filtration with a 0.45
um filter.
a) Most of the charged impurities are usually
anions such as nucleic acids and endotoxins.
These are often separated by ion exchange
chromatography.
b) column chromatography can also use which is
much quicker method.
 Radio immunotherapy (RIT)
Antibody-directed enzyme prodrug
therapy (ADEPT)
 Immunoliposomes
Monoclonal antibodies used for autoimmune
diseases include infliximab and adalimumab,
which are effective in rheumatoid
arthritis, Crohn's disease and ulcerative Colitis.
Basiliximab and daclizumab activated T cells and
thereby help preventing acute rejection of kidney
transplants.
Omalizumab inhibits human immunoglobulin E
(IgE) and is useful in moderate-to-severe
allergic asthma.
Antibodies are used in several diagnostic tests to
detect small amounts of drugs, toxins or
hormones.
e.g. monoclonal antibodies to human chorionic
gonadotropin (HCG) are used in pregnancy test
kits.
Another diagnostic uses of antibodies is the
diagnosis of AIDS by the ELISA test.
Antibody Brand name Type Indication
Abciximab ReoPro chimeric Cardiovascular disease
Adalimumab Humira human Several auto-immune
disorders
Alemtuzumab Campath humanized Chronic lymphocytic
leukemia
Basiliximab Simulect chimeric Transplant rejection
Belimumab Benlysta human Systemic lupus
erythematosus
Bevacizumab Avastin humanized
Colorectal cancer, Age
related macular
degeneration
Brentuximab
Vedotin
Adcetris Chimeric Hodgkin lymphoma
Antibody Brand name Type Indication
Canakinumab Ilaris Human
Cetuximab Erbitux chimeric Colorectal cancer, Head
and neck cancer
Certolizumab
pegol Cimzia humanized Crohn's disease
Daclizumab Zenapax humanized Transplant rejection
Denosumab Prolia , Xgeva Human Postmenopausal
osteoporosis
Eculizumab Soliris humanized Paroxysmal nocturnal
hemoglobinuria
Efalizumab Raptiva humanized Psoriasis
Antibody Brand name Type Indication
Palivizumab Synagis humanized Respiratory Syncytial
Virus
Panitumumab Vectibix human Colorectal cancer
Ranibizumab Lucentis humanized Macular degeneration
Rituximab Rituxan,
Mabthera chimeric Non-Hodgkin
lymphoma
Tocilizumab Actemra Humanised Rheumatoid arthritis
Tositumomab Bexxar murine Non-Hodgkin
lymphoma
Trastuzumab Herceptin humanized Breast cancer

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MCAB

  • 1.
  • 2.  Antibodies  An antibody (Ab), also known as an immunoglobulin (Ig), is a large Y-shaped protein produced by B cells that is used by the immune system to identify and neutralize foreign objects such as bacteria and viruses.  “An antibody produced by a single clone of cells. Monoclonal antibody is therefore a single pure type of antibody”  Each monoclonal antibody recognizes a specific antigen.
  • 3.
  • 4.
  • 5. ANTIBODIES POLYCLONAL MONOCLONAL  Derived from different B Lymphocytes cell lines.  Batch to Batch variation affecting Ab reactivity & treatment.  NOT Powerful tools for clinical diagnostic tests.  Derived from a single B cell clone.  No Batch to Batch variations. Effectiveness of Ab is much more predictable.  Enable the development of secure immunoassay systems.
  • 6.
  • 7.  The types of mAb designed  Murine source mAbs: rodent mAbs with excellent affinities and specificities generated using conventional hydrioma technology. Clinical efficacy compromised by HAMA(human anti murine antibody) response, which lead to allergic or immune complex hypersensitivities.  Chimeric mAbs: chimers combine the human constant regions with the intact rodent variable regions. Affinity and specificity unchanged. Also cause human antichimeric antibody response (30% murine resource)  Humanized mAbs: contained only the CDRs of the rodent variable region grafted onto human variable region framework 
  • 8. 1) Homogeneity: Monoclonal antibody represents a single antibody molecule that binds to antigens with the same affinity and promote the same effectors functions. 2) Specificity: The product of a single hybridoma reacts with the same epitope on antigens. 3) Immunizing Antigen: Need not be pure or characterized and is ultimately not needed to produce large quantities of antibody. 4) Selection: It is possible to select for specific epitope specificities and generate antibodies against a wider range of antigenic determinants. 5) Antibody Production: Unlimited quantities of a single well-defined monospecific reagent.
  • 9. 1) Affinity: Average affinity of monoclonal antibodies are generally lower than polyclonal antibodies. 2) Effector Functions: Because antibody is monoclonal, it may not produce the desired biologic response. 3) Specificity: Monoclonals against conformational epitopes on native proteins may lose reactivity with antigens. 4) Cross reactions: Antibodies sometimes display unexpected crossreactions with unrelated antigens. 5) Time and effort commitment:VERY LARGE.
  • 10.
  • 11.
  • 12.  Process by which bulk quantities of targeted antibodies against a specific antigen are produced.  Monoclonal antibodies are produced via multiple/identical copies of a certain cell called a hybridoma.  To create Hybridoma cells the fusion of 2 cells are needed in order to combine the characteristics of the 2 cells into 1 cell.  1 of the cells is a producing cell antibody which is a B- Lymphocyte used from a laboratory mouse and the other is a tumor cell named myeloma.  Tumor cells have the ability to grow indefinitely and at an exceeding rate from normal cell growth.  Laboratory produced Hybridoma cells replicate much faster than normal antibody producing cells, and the individual hybridomas produce the specific antibodies for an indefinite period of time.
  • 13.  Hybridoma cells manufacture the specific monoclonal antibody that was originally produced by the B- Lymphocyte cell.  The original B-Lymphocyte cell will produce the Monoclonal antibody depending on the kind of antigen that was injected into the mouse just prior to the harvesting of the B-Lymphocyte cells.  Eg: If the mice were injected with a certain virus, the mouse will have B-Lymphocytes that produce those specific virual antibodies. Fusion with a tumor cell to make the hybridoma, result in the production of monoclonal antibodies against the specific virus.
  • 14.
  • 15.  The hybridoma cells are placed into media that can help them grow and produce the bulk quantities of monoclonal antibodies.  There are 2 ways for growing monoclonal antibodies, 1 is to grow them in laboratory flasks meaning In Vitro, and the other is to grow them in the stomach lining of mice.  Injecting the hybridomas into the mice is the familiar method of harvesting monoclonal antibodies. This method is done by mixing spleen cells from the mouse that has been immunized with the desired antigen with myeloma cells. The myeloma cells need to have lost their ability to synthesize HGPRT enzyme (hypoxanthine- guanine-phosphoribosyltransferase) which enables the cells to synthesize purines using an extracellular source of hypoxanthine as a precursor.
  • 16.  Cells have another pathway that they synthesize purines so lack of HGPRT is not a problem for the cell but when cells are exposed to aminopterin they are unable to use this other pathway and are fully dependent on HGPRT for their survival. So to summarize, unfused myeloma cells can’t grow since they lack HGPRT and unfused normal spleen cells can’t grow since they have a limited life-span. Hybridoma cells are grow indefinitely since the spleen- cell copartner supplies HGPRT and the myeloma partner is immortal.
  • 17.  The first step is transferring of the cell fusion mixture to HAT culture medium which contains hypoxanthine, aminopterin & pyrimidine thymidine.  The 2nd step is testing the supernatants from each culture in order to locate the producing the desired antibody.  One must isolate the single cells from each antibody-positive culture and subculture them, this represents the clone which its antibodies are monoclonal.  Every single cell culture secretes a specific kind of antibody that is directed against a certain determinant/selected antigen.  The 3rd step is scaling up the size of the cultures of the successful clones.  Hybridoma cultures can be grown indefinitely in vitro in culture vessels which yield 15-65 µg/ml and in vivo using mouse, where the antibody concentration in the serum 0.5-15 mg/ml.  In the past years, animal welfare activists in worldwide are trying to limit the use of mice for the production of monoclonal antibodies.  When the monoclonal antibody is produced it can be used as a probe to track down, bind to and purify the specific protein that induced its formation.
  • 18. 1) FILTRATION METHOD: Cells, cell debris, lipids, and clotted material are first removed, typically by filtration with a 0.45 um filter.
  • 19. a) Most of the charged impurities are usually anions such as nucleic acids and endotoxins. These are often separated by ion exchange chromatography. b) column chromatography can also use which is much quicker method.
  • 20.
  • 21.  Radio immunotherapy (RIT) Antibody-directed enzyme prodrug therapy (ADEPT)  Immunoliposomes
  • 22. Monoclonal antibodies used for autoimmune diseases include infliximab and adalimumab, which are effective in rheumatoid arthritis, Crohn's disease and ulcerative Colitis. Basiliximab and daclizumab activated T cells and thereby help preventing acute rejection of kidney transplants. Omalizumab inhibits human immunoglobulin E (IgE) and is useful in moderate-to-severe allergic asthma.
  • 23. Antibodies are used in several diagnostic tests to detect small amounts of drugs, toxins or hormones. e.g. monoclonal antibodies to human chorionic gonadotropin (HCG) are used in pregnancy test kits. Another diagnostic uses of antibodies is the diagnosis of AIDS by the ELISA test.
  • 24.
  • 25. Antibody Brand name Type Indication Abciximab ReoPro chimeric Cardiovascular disease Adalimumab Humira human Several auto-immune disorders Alemtuzumab Campath humanized Chronic lymphocytic leukemia Basiliximab Simulect chimeric Transplant rejection Belimumab Benlysta human Systemic lupus erythematosus Bevacizumab Avastin humanized Colorectal cancer, Age related macular degeneration Brentuximab Vedotin Adcetris Chimeric Hodgkin lymphoma
  • 26. Antibody Brand name Type Indication Canakinumab Ilaris Human Cetuximab Erbitux chimeric Colorectal cancer, Head and neck cancer Certolizumab pegol Cimzia humanized Crohn's disease Daclizumab Zenapax humanized Transplant rejection Denosumab Prolia , Xgeva Human Postmenopausal osteoporosis Eculizumab Soliris humanized Paroxysmal nocturnal hemoglobinuria Efalizumab Raptiva humanized Psoriasis
  • 27. Antibody Brand name Type Indication Palivizumab Synagis humanized Respiratory Syncytial Virus Panitumumab Vectibix human Colorectal cancer Ranibizumab Lucentis humanized Macular degeneration Rituximab Rituxan, Mabthera chimeric Non-Hodgkin lymphoma Tocilizumab Actemra Humanised Rheumatoid arthritis Tositumomab Bexxar murine Non-Hodgkin lymphoma Trastuzumab Herceptin humanized Breast cancer