Production of Monoclonal Antibodies and Applications in

therapy and diagnosis

Introduction

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

response to foreign proteins, called antigens. Antibodies function as markers, binding to

the antigen so that the antigen molecules can be recognized and destroyed by phagocytes.

The part of the antigen that the antibody binds to is called the epitope. The epitope is thus

a short amino acid sequence that the antibody is able to recognize.

Monoclonal antibodies (mAb) are antibodies that are identical because they were

produced by one type of immune cell, all clones of a single parent cell.

Characteristics are

(i) Mono-specific - recognize only one epitope (antigenic determinant)

(ii) Homogenous - identical immunoglobulin molecule or Display identical binding

strengths (affinity)

(iii) Produced in unlimited quantities

(iv) Usually derived from mice

Polyclonal antibodies are antibodies that are derived from different cell lines. They

differ in amino acid sequence.

The procedure of production

In 1975, Kohler and Milstein discovered that murine (mouse) antibody-secreting plasma

cells and immortal murine myeloma cells could be fused with the benefits of each

retained. This discovery propelled science and medicine into the modern monoclonal

antibody era.

The basic strategy includes

(i) Purification and characterization of the desired antigen in adequate quantity

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(i) Purification and characterization of the desired antigen in adequate quantity

(ii) Immunization of mice with the purified antigen

(iii) Culture of myeloma cells which are unable to synthesize hypoxanthine-guanine-

phosphoribosyl transferase (HGPRT) enzyme necessary for the salvage pathway

of nucleic acids

(iv) Removal of spleen cells from mice and its fusion with the myeloma cells

(v) Following fusion, the hybridomas were grown in hypoxanthine aminopterin

thymidine (HAT) medium.

Unfused myeloma cells cannot grow because they lack HGPRT

Unfused normal spleen cells cannot grow indefinitely because of their limited

life span.

However, Hybridoma cells (produced by successful fusions) are able to grow

indefinitely because the spleen cell partner supplies HGPRT and the myeloma

partner is immortal.

(vi) The hybrid cell clones are generated from single host cells

(vii) The antibodies secreted by the different clones are then tested for their ability to

bind to the antigen using an enzyme-linked immunosorbent assay (ELISA)

(viii) The clone is then selected for future use

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Applications in therapy and Diagnosis

Monoclonal antibodies are widely used as diagnostic and research reagents as well as in

human therapy. In some in vivo applications, the antibody itself is sufficient. Once bound

to its target, it triggers the normal effector mechanisms of the body.

In other cases, the monoclonal antibody is coupled to another molecule, for example

a fluorescent molecule to aid in imaging the target

a strongly-radioactive atom, such as Iodine-131 to aid in killing the target

Diagnostic application

The diagnostic applications of monoclonal antibodies are by far the most advanced,

especially for tests that are performed on body fluids such as blood and urine sample.

MAb is used to detect pregnancy as early as a week or two after conception by reacting

with human chorionic gonadotrophin, a hormone secreted by the placenta and found in

the urine of pregnant women.

To detect the presence of the substance/antigen, MAbs can be used. Different

technologies in which MAbs are used include Western blot, immunodot blot, ELISA,

radioimmuno assay (RIA), flow cytometry, immunohistochemistry, fluorescence

microscopy, electron microscopy, confocal microscopy, as well as other biotechnological microscopy, electron microscopy, confocal microscopy, as well as other biotechnological

applications. Such as

Gene cloning

One of the difficulties of gene cloning is identifying the cells that contain the desired

gene. If MAb that recognizes that the gene product is available, it can be used as a probe

for detecting those cells that make the product and therapy to detect the gene.

To identify cell types

MAbs contribute to the identification of many different types of cells that participate in

the immune response and to unravel interactions occurring during this process. For

example, in the lymphocytes with B, T helper (TH) cells and suppressor T, the use of

MAbs has established that the various types of T-cells carry cell surface antigens on their

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MAbs has established that the various types of T-cells carry cell surface antigens on their

surfaces that allow one type to be distinguished from another. The MAbs were also

helpful in defining changes in T and B-cells during development.

Protein purification

MAb affinity columns are readily prepared by coupling MAbs to a cyanogen bromide-

activated chromatography matrix, eg, Sepharose. Since the MAbs have unique specificity

for the desired protein, the level of contamination by unwanted protein species usually is

very low. Since the MAb-antigen complex has a single binding affinity it is possible to

elute the required protein in a single, sharp peak. The concentration of the relative protein relative to total protein in a mixture can ever be very low. Therefore Monoclonal

antibodies can also be used to purify a substance with techniques called Immuno-

precipitation and affinity chromatography.

This method also has limitations. Achieving 100% pure protein is difficult because there

is always a tendency for small amounts of immunoglobulin to leak off the immune-is always a tendency for small amounts of immunoglobulin to leak off the immune-

affinity column. Additionally, MAb do not distinguish between intact protein molecules

and fragments containing the antigenic site.

Therapeutic application

Possible treatment for cancer involves monoclonal antibodies that bind only to cancer

cells specific antigen and induce immunological response on the target cancer cell (naked

antibodies). MAb can be modificated for delivery of toxin, radioisotope, cytokine. Toxins

or radioactive isotopes are bound to the constant region of the MAbs. When the MAb

binds to the surface cells of a tumor the toxin or radioactivity will kill the cancer cells and

all cells within a certain radius (a killing zone). In this way cancer cells within the tumor

will be killed.

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Mouse antibodies are "seen" by the human immune system as foreign, and the human

patient mounts an immune response against them, producing HAMA "human anti-mouse

antibodies". These not only cause the therapeutic antibodies to be quickly eliminated from the host, but also form immune complexes that cause damage to the kidneys.

However, using genetic engineering it is possible to make mouse-human hybrid

antibodies to reduce the problem of HAMA. Antibodies with increased efficiency were

engineered for which the murine immunogenic content was removed

Chimeric antibodies: The antibody combines the antigen-binding parts (variable Chimeric antibodies: The antibody combines the antigen-binding parts (variable

regions) of the mouse antibody with the effector parts (constant regions) of a

human antibody. Infliximab, rituximab, and abciximab are examples.

Humanized antibodies: The antibody combines only the amino acids responsible

for making the antigen binding site (the hypervariable regions) of a mouse (or rat)

antibody with the rest of a human antibody molecule thus replacing its own

hypervariable regions. Daclizumab, Vitaxin, Mylotarg, Herceptin, and Xolair

are examples.

In both cases, the new gene is expressed in mammalian cells grown in tissue culture

(i) To suppress the immune system

Muromonab-CD3 (OKT3) and two humanized anti-CD3 monoclonals. Bind to Muromonab-CD3 (OKT3) and two humanized anti-CD3 monoclonals. Bind to

the CD3 molecule on the surface of T cells. Used to prevent acute rejection of

organ, e.g., kidney, transplants. The humanized versions show promise in

inhibiting the autoimmune destruction of beta cells in Type 1 diabetes mellitus.

Infliximab (Remicade) and adalimumab (Humira). Bind to tumor necrosis factor-

alpha (TNF- promise against some inflammatory diseases such as

rheumatoid arthritis (by blunting the activity of Th1 cells). Side-effects: can

convert a latent case of tuberculosis into active disease; can induce the formation

of autoantibodies (by promoting the development of Th2 cells).

(ii) To kill or inhibit malignant cells

Rituximab (trade name = Rituxan). Binds to the CD20 molecule found on most B-

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cells and is used to treat B-cell lymphomas .

(iii) As Angiogenesis Inhibitors

Cancer cells (probably like all tissues) secrete substances that promote the formation of

new blood vessels, a process called angiogenesis.

Vitaxin binds to a vascular integrin (alpha-v/beta-3) found on the blood vessels of

tumors but not on the blood vessels supplying normal tissues. In Phase II clinical

trials, Vitaxin has shown some promise in shrinking solid tumors without harmful

side effects.

(iv) Abciximab (ReoPro). Inhibits the clumping of platelets by binding the receptors on

their surface that normally are linked by fibrinogen. Helpful in preventing reclogging

of the coronary arteries in patients who have undergone angioplasty.

Conclusion

Monoclonal antibodies are monospecific antibodies that are made by identical immune

cells that are all clones of a unique parent cell. Monoclonal antibodies are typically made

by fusing myeloma cells with the spleen cells from a mouse that has been immunized

with the desired antigen. However, recent advances have allowed the use of rabbit B-cells

to form a rabbit hybridoma. Hybridoma a hybrid cell used as the basis for the production

of antibodies in large amounts for diagnostic or therapeutic use. Once monoclonal

antibodies for a given substance have been produced, they can be used to detect the

presence of the substance. The Western blot test and immune-dot blot tests detect the

protein on a membrane. They are also very useful in immunohistochemistry, which detect

antigen in fixed tissue sections and immunofluorescence test, which detect the substance

in a frozen tissue section or in live cells. Therapeutic monoclonal antibodies act through a in a frozen tissue section or in live cells. Therapeutic monoclonal antibodies act through a

number of mechanisms, such as blocking of targeted molecule functions, inducing

apoptosis of cells which express the target, or by controlling signaling pathways.

References

http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/M/Monoclonals.html

http://www.bio.davidson.edu/molecular/MolStudents/01rakarnik/mab.html

http://www.sumanasinc.com/webcontent/animations/content/monoclonalantibodies.ht

ml

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