What is monoclonal antibody 03

Discovery of Monoclonal AntibodiesMonoclonal antibodies were produced in mice using a technique described by Köhler and Milstein et al... Monoclonal antibodies are monospecific antib

Animal Biotechnology

Group 1 05/03/2011

Outline content

• Introduction

• Production of Monoclonal Antibodies

• Advantage and Disadvantages of MAbs

• Application

Georg Kohler and Cesar Milstein fuse mouse

lymphocytes with neoplastic mouse plasma cells

to yield hybridomas that produce specific

antibodies This offers a limitless supply of

monoclonal antibodies Monoclonal antibodies permit diagnostic tests that are specific, and

function as probes

Beginning of Monoclonal Era

Discovery of Monoclonal Antibodies

Monoclonal antibodies

were produced in mice

using a technique

described by Köhler and

Milstein et al They were

awarded the Nobel Prize in

1984 (along with Jerne) for

their work

What is monoclonal antibody

Monoclonal antibodies are proteins that are made in a laboratory

These proteins are designed to attach to areas on the surface of cancer cells and interfere with their growth and spread

Monoclonal antibodies are monospecific antibodies that are identical because they

are produced by one type of immune cell that are all clones of a single parent cell Given (almost) any substance, it is possible to create monoclonal antibodies that specifically bind to that substance

Antibodies are proteins produced by the B lymphocytes of the immune system

in response to foreign proteins, called antigens

Two features of the antibody-epitope relationship are key to the use of

monoclonal antibodies as a molecular tool.

•specificity the antibody binds only to its particular epitope

•sufficiency the epitope can bind to the antibody on its own

Monoclonal antibodies are similar to the antibodies your body naturally

produces when you are exposed to bacteria or viruses, such as a cold or the flu (influenza).

Production of monoclonal antibodies

• Monoclonal antibodies are antibodies produced by one cell

line which are specific to one target (antigen)

• Antibodies derived from mouse hybridomas are of limited use

as human therapeutics, since they produce an adverse

immune reaction with repeated use.

• The term hybridoma is myeloma cell culture applied to fused cells resulting due to fusion of following two types of cells:

1 An antibody producing lymphocyte cell, and

2 A single myeloma cell which is capable of multiplying

indefinitely These fused hybrid cells or hybridoma have the antibody producing capability inherited from lymphocytes and have the ability to grow continuously (immortal) like

malignant cancer cells.

Production of monoclonal antibodies

• Steps in the production of monoclonal antibodies using

hybridoma technology:

1 Immunize a rabbit or mouse 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 From the above two types of animals, culture separately

spleen cells that produce specific antibodies, and myeloma cells that produce tumors.

Production of monoclonal antibodies

4 Induce fusion of spleen cells to myeloma cells, using

polyethylene glycol (PEG), to produce hybridoma; the hybrid cells are grown in selective hypoxanthine aminopterine

thymidine (HAT) medium.

Production of monoclonal antibodies

5 Select the desired hybridoma for cloning and antibody

production.

6 Culture selected hybridoma cells for the production of

monoclonal antibodies in large quantity.

Screening and propagating

• The hybridomas now are ready to be diluted and grown, thus

obtaining a number of different colonies, each producing only one type of antibody.

• The desired antibodies from the different colonies are then

tested for their ability to bind to the antigen (ELISA), and the most effective one is picked out.

ELISA to test Mab production

• The dark blue spot represents a positive clone.

• The light blue spots are positive controls, the next two wells to

the right are negative controls.

Purification of Antibodies

• Before final purification, the cultures may be subjected

to cell fractionation for enrichment of the antibody

protein.

• In E.coli, the antibodies may be secreted in the

periplasm, used for enrichment of antibody, so that

further purification is simplified.

• Alternatively the antibodies may be purified from cell

homogenate or cell debris obtained from the medium.

• Antibodies can be purified by techniques:

1 Ion-exchange chromatography

2 Antigen affinity chromatography

Monoclonal antibodies are produced by

Hybridoma technique

Advantages of Monoclonal Ab

• Single Mabs are chemically defined, are specific

for a particular type of antigen and can be used for standardization of specific assays.

• The Ab created are 100% active antibodies =>

Possible of high specific activity of labelling in

radioimmunoassay, enzyme linked

immunosorbent assay…

• MAbs can be used as immunosorbents for antigen

purification.

Disadvantages of Monoclonal Ab

• MAbs are too specific Limited amounts of MAb migh miss

important cross-reactive determinant.

• Any physical or chemical treatment that affect one molecule

will affect MAbs, causing deactivation of MAbs activities

• Time consuming to produce MAbs, 3-4 months for each fusion

experiment.

Monoclonal antibodies’s

applications

Another diagnostic uses of antibodies

is the diagnosis of AIDS by the ELISA test, diagnosis of hepatitis, influenza, herpes, streptococcal, and Chlamydia infections

- Can be used to detect for the presence and quantity of substances by The Western blot test and immuno dot blot tests.

Therapeutic treatment

• Limitations : The rejection of monoclonal antibodies by the

human immune system

• Solution :

Mice have been genetically engineered to produce antibodies that have human constant regions By using these hybrid, the immune system only "sees" a human protein and does not react against them.

• Cancer treatment :

Monoclonal antibodies bind to cancer cell-specific antigens and induce an immunological

response against the target

cancer cell.

Monoclonal antibodies can also be used as delivery vehicles, guiding radioactive molecules or toxins to the cancer cell.

Antibodies attached to a cell can trigger an immune response that destroys the cell.

Group member

• Phạm Anh Dũng

• Nguyễn Đức Thiện An

• Lê Thị Kim Diệu

• Ngô Huỳnh Thế Hải

• Nguyễn Việt Tuấn