Monoclonal Antibodies

Monoclonal  Antibodies

Antibodies also known as immunoglobulins, are secreted by B cells to destroy antigens such as bacteria, viruses, fungi, toxins and other harmful substances. These antibodies contain paratopes that are specific for particular epitopes found on the antigens allowing both these structures to bind together in a precise way. The ability to bind antibodies to specific antigens has led to their extensive use in a variety of life sciences and medical sciences.

The antibodies produced by plasma cells are classified into two primary types by the sources in which they are synthesized from lymphocytes. Each type has a major role in the immune system.

What are Monoclonal Antibodies?

Antibodies obtained from a single replica of a plasma cell are known as monoclonal antibodies. Monoclonal antibodies have the same antigenic specificity which means they will be produced against a single epitope of an antigen.

Plasma cells producing monoclonal antibodies are always against a specific antigenic determinant. These antibodies are produced by using a technology called hybridoma technology.

The clone of B cells activated against a single antigenic determinant is combined with an immortal cell i.e. myeloma cell. After combination, the hybridoma cell is produced which produces more antibodies.

Monoclonal Antibody Production

The technique for monoclonal antibody production is known as the hybridoma technique which was discovered by Georges J F Koehler and Cesar Milstein in 1975.

The mouse is immunized by specific antigen injection against which monoclonal antibodies are needed to be produced. After 72 hours of immunization spleen is collected from the mouse. (Antibody-producing B cells). Then B cells collected from the spleen are fused with immortalized myeloma cells by polyethylene glycol or Sendai virus. The fused cells are incubated in a HAT medium.

Procedure

Immunization of an animal

The immunization of an animal is the first step in monoclonal antibody production. The mouse is usually used for this purpose. The mouse is immunized with the antigen against which we need monoclonal antibodies. The mouse is injected with the antigen several times. As a result, the mouse B lymphocytes are stimulated against the epitope or antigenic determinants of the antigenic determinants. After 72 hours, when these B lymphocytes reach an optimal amount, the mouse is sacrificed to get a spleen which is a secondary lymphoid organ. The spleen is then subjected to mechanical or enzymatic disruption. This results in the release of cells. Activated B cells or plasma cells are separated from normal spleen’s B cells by density gradient centrifugation. So, the activated B cells are obtained which are capable of producing antibodies against the specific epitopes present on the antigen.

Cell Fusion

B lymphocytes are fused with myeloma cells in this step of hybridoma technology. Myeloma cells are cancerous cells that can divide in a culture but their two genes are mutated. The first one is 8g PRT due to which they are not able to synthesize nucleotides and the second gene is the immunoglobulin gene. As a result of mutation in these genes, the myeloma cells are not able to produce their antibodies.

The cell fusion is done by nixing two types of cells in the presence of polyethylene glycol. By fusion, five types of cells are obtained which are unfused B cells, fused B cells, unfused myeloma cells, fused myeloma cells and hybrid cells formed by the fusion of activated B cells and myeloma cells. These hybrid cells are also called hybridomas.

Selection of Hybridomas

The hybridoma cells are selected by using a HAT medium. The combination of cells picked up after the fusion of cells is passed on to the HAT medium. HAT stands for Hypoxanthine, Aminopterin and Thymidine. HAT medium is a selection medium from the mammalian cell cultures. The fused and unfused B cells will die within a few days because of their short lifespan. The fused and unused myeloma cells will also die because the myeloma cells are 8g PRT negative and Aminopterin present in the HAT medium that blocks the de Novo pathway. The hybridoma cells can be synthesized in a HAT medium because they can produce nucleotides by the salvage pathway. The 8g PRT is contributed by the B cells and the ability to divide indefinitely is due to myeloma cells. Therefore, the hybrid cells survive in the HAT medium, which promotes the production of antibodies against the specific epitope of an antigen. Now the hybrid cells are again a mixture of B cells producing antibodies of different specificities. Each B cell will synthesize antibodies specific to different epitopes on the same antigen.

Isolation of Hybridomas of Single Specificities

the hybridomas of single specifies are isolated by limiting dilution. The hybridoma cells are distributed in multi well cultures at a very low density. This is done in such a way that each culture will contain a single cell.

Screening of Products

In this step, the hybridomas are screened for the secretion of antibodies of desired specificities. The two mostly commonly used techniques for this purpose are ELIZA and RIA.

Cloning and Propagation

Once the hybridoma cells producing the desired antibodies are identified, they are isolated and cloned in the next step. These antibodies are monoclonal antibodies with a single specificity.

Characterization and Storage

In the final step, the hybridomas and monoclonal antibodies are characterized and stored in liquid nitrogen. These antibodies are ready to use for treating and diagnosing diseases.

Applications of Monoclonal Antibodies

Biochemical Analysis

Monoclonal antibodies are routinely used in radioimmunoassay (RIA) and enzyme-linked immunosorbent assays (ELISA) in the laboratory which measure the circulating concentrations of hormones (insulin, human chorionic gonadotropin etc.) and several other tissue and cellular products (blood group antigens, blood clotting factors, interferons, and tumor markers).

Diagnostic Imaging

Radiolabeled monoclonal antibodies are used in the diagnostic imaging of diseases, and this technique is referred to as. The Immunoscintigraphy. The radioisotopes commonly used for labeling monoclonal antibodies are Iodine-131 and Technetium-99. The monoclonal antibodies tagged with a radioisotope are injected intravenously into the patients. These monoclonal antibodies localize at specific sites (say a tumor) which can be detected by imaging the radioactivity e.g., myocardial infarction, atherosclerosis, etc.

Therapeutic Agents

1. In the treatment of cancer: Monoclonal Antibodies, against the antigens on the surface of cancer cells, are useful for the treatment of cancer. The antibodies bind to the cancer cells and destroy them via different pathways.
2. In immunosuppression of organ transplantation: In normal medical practice, immunosuppressive drugs such as cyclosporine and prednisone are administered to overcome the rejection of organ transplantation. In recent years, Monoclonal antibodies specific to T-lymphocyte surface antigens have been used for this purpose.
3. In destroying disease-causing organisms: Monoclonal antibodies promote efficient opsonization of pathogenic organisms (by coating with antibodies) and enhance phagocytosis.

 As targeting Agents

The medicines can be fused with monoclonal antibodies and especially selected to reach the site of action. For example:

• Alkaline phosphatase for the changing of phosphate pro-drugs.
• Carboxypeptidase for converting inactive carboxyl pro-drugs to active drugs.
• Lactamase for the hydrolysis of the ß-lactam ring-containing antibiotics.

For Protein Purification

Monoclonal antibodies can be produced for any protein and the so produced antibodies can be conveniently used for the purification of the protein against which it was raised.

Monoclonal antibody columns can be prepared by coupling them to cyanogen bromide-activated Sepharose (chromatographic matrix). The immobilized monoclonal antibodies in this manner are very useful for the purification of proteins by the immunoaffinity method.

There are certain advantages of using monoclonal antibodies for protein purification. These include the specificity of the monoclonal antibodies to bind to the desired protein, very efficient elution from the chromatographic column and a high degree of purification.