Download Cell identification using Monoclonal Antibodies

Cell identification using
Monoclonal Antibodies
What are Monoclonal Antibodies
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Monoclonal antibodies (MAbs) are:
 antibodies of exceptional purity and specificity
 components of the immune system
 able to recognize and bind to a specific antigen
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In 1975, Kohler and Milstein first fused lymphocytes to produce a cell line
which was both immortal and a producer of specific antibodies. The two
scientists were awarded the Nobel Prize for Medicine in 1984 for the
development of this "hybridoma." The value of hybridomas to the field was not
truly appreciated until about 1987, when MAbs were regularly produced in
rodents for diagnostics.
polyclonal antiserum vs. monoclonal antibodies
Production of Mab
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Technology: The first step in the production of MAbs is to immunize a mouse
with an antigen. When the mouse begins to produce antibodies to the antigen,
its spleen is removed. Antibody-producing cells from the spleen are then fused
with a selected myeloma cell line, one which is not Ab-producing and has been
selected to be deficient in an enzyme in the nucleotide salvage pathway
(hypoxanthine-guanine phosphotransferase, HGPRT).. The new fused cell line,
which does produce antibodies, can be grown in culture or re-injected into
another mouse's peritoneum.
Nucleotide synthesis
Production of
Monoclonal Abs
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Principal Application of Monoclonal Abs
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Identification, enumeration, localisation and isolation
of individual types of cells from solid tissue or blood
or from any microorganisms.
 Molecules expressed selectively by a particular
type of cell is selected as a marker
 Monoclonal antibodies against such a marker can
be used to identify, count, find and isolate cells
carrying the marker.
Some Uses of Monoclonal Antibodies
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Monoclonal antibodies are currently utilized in many diagnostic
procedures, including:
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measuring protein and drug levels in serum
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typing tissue and blood
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identifying infectious agents
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identifying clusters of differentiation for the classification and
follow-up therapy of leukemias and lymphomas
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identifying tumor antigens and auto-antibodies
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identifying the specific cells involved in the immune response
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identifying and quantifying hormones
Techniques Used
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Identification, enumeration ( if standard present)
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ELISA
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Agglutination
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Biotin-avidin immunodetection
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Western-blotting
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Radioimmuno assay
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Radioimmuno guided surgery
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Eg:I125 labeled anti-TAG72 Ab in detection of colorectal tumours
Physical process to separate cells
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Magnetic beads
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FACS
Properties and Uses of ELISA
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ELISA is a very sensitive assay
The assay is easily automated
Can be used to quantitate antigen or antibody
Typically used for immuno-diagnosis of many bacterial, viral
and parasitic infections.
The standard screening test for HIV is the ELISA. However,
there are many false positives, thus, a positve ELISA must be
confirmed by a more specific assay. The most commonly
used confirmatory test is the western blot.
Enzyme-Linked Immunosorbent Assay
ELISA
Western Blotting
Cellular proteins reacting with an antibody can be characterized
by immunoprecipitation of labeled cell lysates
Agglutination
Bacterial Agglutination
Agglutinated
Non-agglutinated
Hemagglutination: Blood group identification
Blood groups
Radioimmuno Assay
The 125I is measured between blank and sample
Immunological Cell Separation
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Rare cell isolation:
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Stem cells (CD34+, AC133)
Natural killer cells (CD56+)
Cancer cells circulating in the blood
 1 in 106 or less?
Foetal cells in maternal blood
Harmful Bacteria (O-157 etc)
Cell Separation Technologies
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Centrifugation : Density
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Filtration : Size
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Flow Cytometry (FACS)
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Size
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Granularity
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Fluorescence
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Batch affinity systems : antibody, avidin-biotin
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Batch magnetic systems
How can cells be separated magnetically?
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Cells are incubated with an antibody specific for a particular cell
surface epitope
The antibody is linked to a magnetic bead (one step labelling), or a
secondary reagent linked to a magnetic bead is added that reacts
with the first antibody (2 step labelling).
These labelled cells are then flowed over a column consisting of a
paramagnetic matrix and exposed to a strong magnetic field.
Unbound cells are eluted, then the magnet is turned off and the
specific cells are eluted.
Immunomagnetic Labeling of Cells
Target antigen
Antibody (IgG)
Cell
Cell
FITC
Magnetic bead
One-Step
Two-Step
Labeling
Labeling
Magnetic-Bead Cell separation system
Plunger
Magnet
Separation
column
Remove the magnet
magnetic cell
non-magnetic cell
Unlabeled
cells
Immunomagnetically
labeled cells
Subpopulations of cells can by physically separated using antibodies coupled
to magnetic beads
Separation using Magnetic beads