monoclonal antibodies

When an antigen is introduced into the circulatory system of a higher vertebrate, it stimulates specific B-lymphocytes to produce antibodies. The antibodies are the immunoglobulins and they circulate in the blood serum.

When the immune system of an animal encounters a new antigen, it responds to specific antigenic determinants called 'epitopes' located on the antigen. Thus, a protein antigen may possess several epitopes and would induce the formation of several different antibodies, each specific for one epitope. Such a polyclonal antibody response facilitates the localization, phagocytosis and complement-mediated lysis of antigens.

For most reasearch, diagnostic and therapeutic purposes, "Monoclonal antibodies", derived from a single clone and thus specific for a single epitope are preferable.

In 1975, "Georges Kohler" and "cesar Milstein" devised a method for preparing monoclonal antibody, which quickly became one of the immunology's key technologies. These work got Nobel Prize in 1984.

Basic concept Principle on the monoclonal antibodies production:

By fusing a normal activated, antibody producing B-cell with a myeloma cell(a cancerous plasma cell), they were able to generate a hybrid cell, called a 'hybdridoma', that possessed the immortal-growth properties of the myeloma cell and secreted the antibodies produced by the B-cell. The resulting clones of hybridoma cells,which secrete large quantities of monoclonal antibodies can be cultured indefinitely. The development of techniques for producing monoclonal antibody gave immunologists a powerful and versatile research tool.

Production &Selection of hybridoma cells:

One common method requires the use of myeloma cells that are deficient (because of previously selected mutation) for one of the nucleotide salvage pathways, making them unable to grow in HAT medium (named for its three components -Hypoxanthine, Aminopterin, and Thymidine). If the mixture of hybridomas and unfused parental cells is placed in this medium, the parental myeloma cells can not survive. The B-cell X myeloma cells can survive because the B-cell contributes the missing enzyme for the salvage pathway. Although unfused B-cells are able to survive in HAT medium, these cells do not live for extended periods in vitro and thus die out.

HAT selection depends on the fact that mammalian cells can synthesize nucleotides by two different pathways; the denovo and the salvage pathways. The denovo path way, in which a methyl or formyl group is transferred from an activated form of tetrahydrofolate, is blocked by Aminopterin, a folic acid analogue. When the denovo path way is blocked, cells utilize the salvage pathway, which bypasses the aminopterin block by converting purines and pyrimidines directly intoYiucleotides for synthesis of DNA and RNA. The enzymes catalyzing the salvage pathway include Hypoxanthine-Guanine Phosphoribosyl transferase(HGPRT) and Thymidine kinase(TK). A mutation in either of these two enzymes blocks the ability of the cell to use the salvage pathway. HAT medium contains aminopterin to block the denovo pathway and hypoxanthine and thymidine to allow growth by the salvage pathway. Therefore, cells that lack either HGPRT or TK will die in HAT medium, because they lake the ability to use the salvage pathway to acquire essential intermediates for the synthesis of nucleic acids.

In hybridoma technology, the myeloma cells are actually double mutants. As mentioned above, they lack the enzyme HGPRTase and therefore are deselected in HAT. They have lost the ability to produce immunoglobulins (Ig- mutants).

Applications of Monoclonal Antibodies:

1. Improved diagnostic reagents: For the assay of wide range of compounds, including hormones, antibiotic interferons, and blood-clotting factors, antibody-antigen reactions are widely used. For blood typing and diagnostic microbiology, the ability of antibodies to agglutinate or precipitate cells have been used; the homogeneity of Monoclonal antibodies reduces reaction times and the likelihood of non-specific cross-reactions between the antibody and antigen is lessened by the use of Monoclonal antibodies. It is possible to diagnose pregnancy or ovulation more precisely with the Monoclonal antibodies.

2.Protein purification: Monoclonal antibody affinity columns are prepared by coupling monoclonal antibodies to a cyanogen bromide-activated chromatography matrix, for example, sepharose. Monoclonal antibodies immomabalized in this way are particularly valuable for the purification of proteins. Since the monoclonal antibodies has a unique specificity for the desired proteins, the level of contamination by unwanted proteins, usually, is very low. Even when the concentrating of the desired protein, in a mixture of proteins, is very low, Monoclonal antibodies has the capacity to combine with it and to remove the whole of it. For example, when the concentration of interferon was less than 0.02%, the anti-interferon monoclonal antibodies enable the recovery of 97% of the interferon by immunoaffinity chromatography.

3.Improved sensitivity and reproducibility of existing immunoassays or new assays for

Histocompatibility antigens, Complement component, Human growth hormones, Interkeukins, Oestrogen, Blood clotting factors, Sperm antigens, Blood group antigens, Progesterone, Fibronectin, Interferons, Gastrin

4. Therapy:

• Correction of drug over dose
• Reduction of risks associated with bone marrow transplants
• Detection of tumour metastases
• Treatment of cancer (directly, or by targeting “cytotoxic drugs”.

5. Diagnosis of:

• Sexually Transmitted Diseases (STDs)
• Cancer(by detection of Onco -foetal antigens)