Microbiolgy Immunology

2. MONOCLONAL ANTIBODY=singal type of antibody
,directed against specific epitope.
POLYCLONAL ANTIBODY=in response to an
antigen(with several different epitopes),B cells
gear up and produce many different
antibodies.this type of antibody which can react
with same antigen are called as polyclonal
antibodies.

3.  B-CELLS+MYLEOMA=HYBRIDOMA
andThe production of monoclonal antibodies
by the hybrid cells is referred to as
hybridoma technology.

4. Georges Kohler
CesarMilstein
Niels Jerne
The production of monoclonal
antibodies was invented by
Cesar Milstein and Georges
Kohler in 1975.they shared the
nobel prize of 1984 for medicine
and physiology with Neils Jerne,
who made other contribution to
immunology and the term
hybridoma was coined by
leonard herzenberg in Cesar
Milstein’s laboratory in
1976/1977

5. Antibody producing lymphocyte B cell are isolated from the
spleen cell of mouse immunized against a specific antigen
Single myeloma cell is a bone marrow tumour cells of
multiplying indefinitely.
Fused hybrid cells or hybridoma have the antibody producing
capability inherited from lymphocytes and have the ability to
grow continuously or immortal like malignant cancer cells.

6. 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

7. 1. Immunize a rabbit through repeated
injection of a specific antigen for the
production of specific antibody, facilitated
due to proliferation of the desired B cells.
2. Produce tumors in a mouse or a rabbit.
3. Culture separately the spleen cells that
produce specific antibodies and the
myeloma cells that produce tumors
4. Myeloma cells cannot synthesize antibodies
as they lack HGPRT gene required for the
synthesize the enzyme, hypoxanthine
guanine phosphoribosyl transferase

8. • Fusion of spleen cells to myeloma cells is
induced using polyethylene glycol (PEG), to
produce hybridoma
• Hybridomas are grown in selective
hypoxanthine aminopterin thymidine (HAT)
medium.
• HAT medium contains a drug, aminopterin
that blocks one pathway for nucleotide
synthesis, making the cells dependent on
another pathway that needs HGPRT enzyme,
which is absent in myeloma cells.
• Myeloma cells that do not fuse with B cells
will die.
• B cells that do not fuse will also die because
they lack tumorigenic property of immortal
growth.

10.  Select desired hybridoma for cloning and antibody
production
 Prepare single cell colonies that can grow and use
them to screen of antibody producing hybridomas
 Only one in several hundred cell hybrids will produce
antibodies of the desired specificity
 Culture selected hybridoma cells for the production of
monoclonal antibodies in large quantities
 Hybridoma cells can be frozen for future use or can be
injected in the body of an animal, so that antibodies
will be produced in the body and recovered later from
the body fluid.

11. Monoclonal antibody truely represent a
homogeneous state of a single molecular
species.
Each monoclonal antibody is specific for a
given antigenic determinant
and there are wide range of aaplications of
monoclonal antibodies.
It is possible to select for specific epitope
specifities and generate antibodies against a
wider range of antigenic determinants.

12.  Hybridoma technology is laborious and time
consuming.
 Mabs are produced against a single antigenic
determinant ,hence they do not differentiate the
molecule as a whole . Sometime they may be
incapable of distinguishing groups of different
molecule also.
 Mice used in Mab production carry several
viruses(adeno viruses ,hepatic viruses).the
presence of these viruses has been detected in
hybridomas.this posses a great danger there is
no guarantee that Mab is totally virus free,
despite the purification .

13.  Diagnosis of HIV virus
 The test of HIV
infection is based on
detecting the presence
of HIV antibody in the
patient’s blood serum

14. a) HIV antigen is attached to the plate.
b) Patients serum passed over the plate. Any HIV
antibody in the patients serum will attached to the
antigen already on the plate.
c) A second antibody which is specific to the HIV
antibody is passed over the plate. This antibody
will attach to the concentrated HIV antibody on
the plate. This second antibody has an enzyme
attached to its structure.
d) Chromagen dye is passed over the complex of
concentrated HIV antibody/conjugated antibody.
e) The enzyme will turn the chromagen to a more
intense colour. The more intense the colour, the
greater the HIV antibody level. This would be the
a positive result for a HIV test.

15.  Identification of ABO blood groups.
 Detection of pregnancy by assaying of
hormones with monoclonals,antibodies.
 Detection of pathogen.
 Separation of one substance from a mixture
of very simmilar molecules .
monoclonal antibodies are used in the
diagnostic immaging of diseases,and this
technique is referred as immunoscintigraphy.

16. Monoclonal antibodies use as immunotoxins-
toxins +Mabs=immunotoxins ,used in therapy.
RICIN- derived from castor oil plant,composed
of two polypeptide chains( A and B),held
together by disulfide linkage.
The B chain of ricin bind to the cell
surface.this binding facilitates the A chain of
ricin to enter the cell and inhibit the
function of ribosomes(i.e. Biosynthesis of all
protein is blocked).this result in death of
cells.

17.  RICIN can be subjected to oxidation to
separate to A and B chains. The toxic A chain
can be conjugated to Mab that is specific to
cancer cells.
 The tumor specific Mab bound to A chain of
ricin binds to cancer cells and not to normal
cells . Once the A chain enters the cells ,it
blocks ribosomal functions leading to the
death of cancer cells.

19.  Monoclonal antibody in drug delivery-the drugs can
be coupled with monoclonal antibody(directed
against a cell surface antigen of the cells,say a tumor
) and specifically targetted to reach the site of
action.
 In the treatment of certain diseases ,a prodrug(an
inactive form of drug)can be used.this can be
enzymatically converted to active drug inthe target
tissues.the enzyme that convert prodrug to drug is
coupled with Mab.that is directed against a specific
cell surface antigen this approach is referred as
antibody directed pro drug therapy(ADEPT).e.g
alkaline phosphatase used for the conversion of
phosphate drugs.

21. A great majority of natural death are due to a
blockage in coronary or cerebral artery by a
blood clot (Thrombus).fibrin is a major
constituent of blood clot which get dissolved
by plasmin.
Plasmin in turn is form by the activation of
plasminogen by plasminogen activator.
monoclonal antibody directed against fibrin
can be coupled to tissue plasminogen
activator and used for degradation of blood
clot .

23.  The radioisotope can be coupled to monoclonal
antibodies that ae directed againsttumor cells this
allows the cocentration of radioactivity at desired
site and very efficient killing of target cells (tumor
cells).
 The advantage with radioimmunotherapy is that
conjugated complex need not penetrate the cells,as
is required in immunotoxin therapy disadvantage is
that neighbouring normal cells also get damaged or
killed.
 This can be minimised by using radioisotope with
short half lives .yttrium -90 with a half life span of
64hrs. Due to shortage in supply of yttrium,indium
111 is more commonly used and they should have half
life and decay bi-product should be inert.

24.  By using affinity chromatography technique
Mabs can be used for the purification of
protein e.g.fetoprotein and interferon.
 Identification of previously unknown antigen.
 Fluorescent labelled Mabs are used for
identification ,isolation of cells displaying a
specific antigen on their surface.
 Mabs have been used to isolate mRNAs
encoding the protein to which Mabs are
specific.

25.  The antibody enzymes ,appropriately
regarded as abzymes ,are catalytic
antibodies.
 It should be possible to generate such
abzymes that catalyze reactions for which no
natural enzyme exist.