Applications and

Uses of
Monoclonal
Antibodies
N.H.K.S.Senathilake
Student Index no.8558
Department of Chemistry
University of Colombo
Sri lanka.

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Applications and Uses of Monoclonal
Antibodies
Immunoassay(ELISA & RIA)
Therapeutics
Antibody micro array
T Immunocytochemistry
H Immunoflourescent
Research R
Flowcytometery
O
Immuno elecctron
U microscopy

Diagnostics G Immunoprecipitation
H
immunoagglutination

Immunoblotting
Immuno purification
07/31/09 Immunoaffinity Purification 2
Applications are coming
through
 There are a large range,these include:
 Immunoassay(ELISA & RIA)
 Antibody micro array
 Immunocytochemistry ( )

Immunoflourescent
 Flowcytometery
 Immuno elecctron microscopy
 Immunoprecipitation
 immunoagglutination
 Immunoblotting
 Immunoaffinity Purification

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Applications of mAbs…….
 Immunoassay
 A factor is quantified on the binding of
specific Ab. The Ab is labeled with something
that can be measured and then compared to a
calibration curve.
 Labels include Enzymes, Isotopes or
Fluorescent tags.
 For an assay to be successful it needs to be:
1. highly sensitive
2. have low non-specific binding
3. able to assay a large number of samples in
at a time
 Widely used examples include ELISA and RIA.

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 Applications of mAbs
RIA
Here antibodies that are specific to
an antigen is coated on wells.

Initially wells are washed with radio

labeled antigens

Then they are washed with test

sample.
Difference of radioactivity can
correlate with amount of test antigens
using a standard plot.

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Applications of mAbs
Enzyme linked immunosobant assay(ELISA)

ELISA (Enzyme Link

Immuno Sorbent Assay )

Antibodies are initially

washed with test sample.

After that wells are

washed with enzyme
linked labeled
secondary a Enzyme
can covert antibodies.
a chromogenic substrate to a colored
product.
So using a ELISA plate reader
we can quantify antigens.

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 Applications of mAbs
 Antibody micro array
 Analyses expression
levels of proteins in
disease diagnosis

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Applications of mAbs
 Antibody micro array

c T

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Applications of mAbs
 Immuno fluvorescent & Immuno electron microscopy
labeled Ab which binds specifically to a molecule of
interest in a fixed cell or tissue mounted on a
microscope slide.
 Label used
Fluorescent tag in immuno fluorescent
Electron dense label like Ag in IEM

Immuno fluorescent Immuno electron

micrograph.
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Applications of mAbs
Immunocytochemistry
 Immunocytochemistry & flowcytometry

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 Applications of mAbs
 Flow Cytometry
Separate and quantify different types
of cells based on difference of
Surface antigens

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Applications of mAbs

 Immunoblotting(western blotting)
 This process involves the characterisation of a factor based
on the MW and ability of Ab to bind to it.
 It is used to identify and characterise antigens from a complex
mixture.
 The antigen samples are resolved by separation in an analytical
gel such as dodecyl sulphate (SDS), peptide mapping gels or
isoelectric focusing gels.
 The resolved molecule are transferred electrophoretically to a
nitrocellulose membrane in a blotting tank.
 The blot is then treated with a MAb to the specific antigen in
question.
 it is then washed and treated with a radiolabel led conjugate,
which detects the bound antibodies.
 This is then washed again and put in contact with a X-ray film to
produce a autoradiograph.
 The bands of antigen that have been bound by the MAbs are
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visible.
Applications of mAbs

 Immunoaffinity Purification
 In this process an antibody on a column
isolates a factor from a mixture. It allows the
purification of a substance from 1,000 fold to
10,000.

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 Applications
Basically, two areas are developed using
above principals that are used in a clinical
laboratory.
Diagnostic

Therapeutic

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Diagnostic
principle applied diagnosis
 ELISA  human and animal
bacterial and
mycoplasmal disease
 Immunocytochemistry
 Leukemia
immunofluoroscence
 Radio immuno assay  Ovarian cancer
 immunofluoroscence diagnosis

 Sexually transmitted
diseases
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Diagnostic (ctd)

 Immunocytochemistry  Allied functional

& immuno electron bowel disoders
microscopy

 Dot-ELISA  Human leptospirosis

 Agglutination
 Pregnancy testing

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 Therapeutic

Immunotoxin Antibody engineering

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Immunotoxins
 Immunotoxins are valuable therapeutic reagent.
 It is composed of tumor-specific monoclonal Ab
coupled to lethal toxins.
Eg:diphtheria,shigella
 Immnotoxins specific for a particular tumor cell &
attach to tumor specific antigen.
 MAb deliver toxin chain into cytosol.
 It will cause death by inhibiting protein synthesis.

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Antibody engineering


Earlier used mouse MAb for clinical applications.
 But arised certain problems
• Allergic reactions
• Life-threatening reaction
 To overcome this problem use human MAb
• Difficult to produce MAb

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Chimeric Antibody

 Engineer MAb & antibody binding sites with

recombinant DNA technology
 Chimeras may be able to deliver powerful
toxin to particular targets such as tumor cells.
Eg:chimaric human mouse Ab used to
treat patients with B-cell varieties of non
Hodgkin's lymphoma.

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 Qestions?

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