Gr 12 Monoclonal Antibodies

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Fusion of a tumor cell with an antibody-producing cell creates a hybridoma cell
Monoclonal antibodies are produced by hybridoma cells

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Procedure for Monoclonal Antibodies

Monoclonal antibodies are antibodies artificially derived from a single B cell clone (i.e. identical specific antibodies)

An animal (typically a mouse) is injected with an antigen and produces antigen-specific plasma cells
The plasma cells are removed and fused (hybridised) with tumor cells capable of endless divisions (immortal cell line)
The resulting hybridoma cell is capable of synthesising large quantities of monoclonal antibody

Production of monoclonal antibodies

A primary immune response by an organism is called a polyclonal response. This is because the pathogen is typically being recognized as many antigens and not just one. For example, the capsid (protein coat) of a virus is made up of several different kinds of protein. Each of the protein types can cause an immune response, and thus several different kinds of plasma B cells undergo clonal selection, so several different kinds of antibodies are produced. Once a polyclonal immune response has occurred, it is very dif cult to separate the different kinds of antibodies that have been produced. Researchers have developed a clever and unique procedure for forming many antibodies all of the same type. These ‘pure’ antibodies are called monoclonal antibodies.

The procedure for producing monoclonal antibodies begins with the injection of an antigen into a laboratory animal such as a mouse. The choice of the antigen is very important because the antibodies that will be produced will bind only to this specific antigen. After the injection, the animal is given time to go through a primary immune response. After an appropriate period of time, the spleen of the animal is ‘harvested’ in order to obtain many blood cells. At least some of the leucocytes cloned for the antigen that was recently injected will be a part of the cellular population within the spleen.

There are two problems that need to be addressed at this point in the procedure:

• keeping the B-cell types alive for an extended period of time

• identifying the B-cell type that produces the antibody that recognizes the desired antigen.

The B cells are kept alive by fusing them with cancerous (myeloma) cells. When B cells and myeloma cells are grown together in the proper environmental conditions, a few of the cells fuse together and become a hybrid cell called a hybridoma. These hybrid cells have characteristics of both cells: they produce antibodies of a particular type and they are very long-lived (as are all cancer cells). The entire mix of cells is now transferred to an environment in which only the hybridoma cells can survive, and all of the B cells and myeloma cells that did not fuse die. Individual surviving hybridoma cells are now cultured in separate containers. Each container is tested for the presence of a particular antibody. The typical protocol to test for a speci c protein (such as an antibody) is called an enzyme-linked immunosorbent assay (ELISA). An ELISA identifies which containers hold a pure colony of B cells that are producing the type of antibody desired. These cells can be cultured for a very long period of time because they have some of the characteristics of a tumour cell. In other words, the hybridoma cells are virtually immortal as long as they are kept in a suitable environment.

Monoclonal antibodies can be used for both the therapeutic treatment and clinical detection of disease

An example of therapeutic use involves the use of antibodies in the treatment of rabies
An example of diagnostic use involves the use of antibodies in the detection of pregnancy

Treatment Use

Monoclonal antibodies are commonly used to provide immune protection for individuals who contract harmful diseases

Because the rabies virus can potentially be fatal, injecting purified antibodies functions as an effective emergency treatment
Monoclonal antibodies can be used to target cancer cells that the body’s own immune cells fail to recognise as harmful
They can be used to locate cancer cells, which have proteins in their cell surface membranes that differ from the protiens on normal body cells and can therefore be detected by antibodies
They can also be used to identify the exact strain of a virus or bacterium that is causing an infection; this speeds up the choice of the most appropriate treatment for the patient.
Mabs are also used routinely in blood typing before transfusion, and tissue typing before transplants.

Therapeutic monoclonal antibodies are named according to the source organism from which the antibodies were derived

Mice antibodies ('-omab’) are easier to synthesise than human antibodies but are less likely to be tolerated by the patient

The antibodies are produced by mice, rabbits or other laboratory animals. When introduced into humans, they trigger an immune response because they are foreign (non-self) and act as antigens. This problem has now been largely overcome by humanising Mabs in two ways:

■ altering the genes that code for the heavy and light polypeptide chains of the antibodies so that they code for human sequences of amino acids, rather than mouse or rabbit sequences (Figure Below)

■changing the type and position of the sugar groups that are attached to the heavy chains to the arrangement found in human antibodies.

Pregnancy Tests

Monoclonal antibodies can be used to test for pregnancy via the presence of human chorionic gonadotrophin (hCG) in urine

hCG is a hormone produced by women during foetal development and thus its presence in urine is indicative of pregnancy

Pregnancy tests use a process called ELISA (enzyme-linked immunosorbent assay) to identify a substance via a colour change

Free monoclonal antibodies specific to hCG are conjugated to an enzyme that changes the colour of a dye
A second set of monoclonal antibodies specific to hCG are immobilised to the dye substrate
If hCG is present in urine, it will interact with both sets of monoclonal antibody (forming an antibody ‘sandwich’)
When both sets of antibody are bound to hCG, the enzyme is brought into physicial proximity with the dye, changing its colour
A third set of monoclonal antibodies will bind any unattached enzyme-linked antibodies, functioning as a control

How does a pregnancy test work? (using monoclonal antibodies)

Monoclonal antibodies can be used for a wide variety of diagnostic purposes. One common use is pregnancy testing. Early in pregnancy, the embryo begins to produce a hormone called human chorionic gonadotropin (HCG). Because HCG is a hormone produced by the embryo, only pregnant women have this hormone; the hormone shows up in small amounts in the bloodstream and urine. Hybridoma cells can be formed that produce antibodies specific for HCG. These anti-HCG antibodies are chemically bonded to an enzyme that catalyses a colour change when the antibody encounters HCG molecules. This is why pregnancy test results involve a colour indicator.

ELISA Assay and Antibodies

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