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

Research

Diagnostics

T H R O U G H

Immunocytochemistry Immunoflourescent Flowcytometery Immuno elecctron microscopy Immunoprecipitation immunoagglutination Immunoblotting Immunoaffinity Purification 2

Immuno purification
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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…….
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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
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.

Enzyme linked immunosobant assay(ELISA)

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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

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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
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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 07/31/09 12 visible.

Applications of mAbs
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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

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Immunocytochemistry immunofluoroscence Radio immuno assay immunofluoroscence

human and animal bacterial and mycoplasmal disease Leukemia Ovarian cancer diagnosis Sexually transmitted diseases
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Diagnostic (ctd)

Immunocytochemistry & immuno electron microscopy

Allied functional bowel disoders

Dot-ELISA Agglutination

Human leptospirosis

Pregnancy testing

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Therapeutic

Immunotoxin

Antibody engineering

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Immunotoxins
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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
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Earlier used mouse MAb for clinical applications. But arised certain problems
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Allergic reactions Life-threatening reaction Difficult to produce MAb

To overcome this problem use human 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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