Monoclonal antibodies are identical immunoglobulins, generated from a single B-Cell clone. These antibodies recognize unique epitopes or binding sites on a single antigen. Derivation from a single B-Cell clone and subsequent targeting of a single epitome is what differentiates monoclonal antibodies from polyclonal antibodies.
Polyclonal antibodies are antibodies that are derived from different cell lines. They differ in amino acid sequences.
Characters of Monoclonal Antibodies
- Monoclonal Antibodies (mAB) are a single type of antibody that are identical and directed against a specific epitope (antigen, antigenic determinant) and are produced by B-Cell clones of a single parent or a single hybridoma cell line.
- A hybridoma cell line is formed by the fusion of one B-cell lymphocyte with a myeloma cell.
- Some myeloma cell synthesizes single mAB antibodies naturally.
Advantages of Monoclonal Antibodies
- Though expensive mAB is cheaper to develop than conventional drugs because it is based on tested technology.
- Side effects can be treated and reduced by using mice-human hybrid cells or by using fractions of antibodies.
- They bind to specific diseased or damaged cells needing treatment.
- They treat a wide range of conditions.
Disadvantages of Monoclonal Antibodies
- A time-consuming method as it requires an average of 6-9 months.
- It is very expensive and needs considerable effort to produce them.
- Small peptide and fragment antigens may not be good antigens-monoclonal antibodies may not recognize the original antigen.
- Hybridoma culture may be subject to contamination.
- The system is only well developed for limited animals and not for other animals.
- More than 99% of the cells do not survive during the fusion process-reducing the range of useful antibodies that can be produced against an antigen.
- It is every possibility that immunogenicity can be generated.
Preparation of Monoclonal Antibodies
- Monoclonal Antibodies Products (mAB) are produced by cells lines or clones obtained from the immunized animals with the substances. Cell lines are produced by fusing B-cells from the immunized animal with myeloma cells.
- To produce the desired mAB, the cells must be grown in either of two ways: 1. By Injection into the peritoneal cavity of a suitably prepared mouse (in vivo method). 2. In vitro Tissue Culture.
- The vitro tissue culture is the method used when the cells are placed in a culture outside the mouse, the mouse’s body in the flask.
Practical Steps for Production
- Immunize animal.
- Isolate spleen cells (containing antibody-produced B-cell).
- Fuse spleen cells with myeloma cells (using PEG).
- Allow infused B-cells to die.
- Add aminopterin to culture and kill unfused myeloma cells.
- Clone remaining cells (place 1 cell/wall and allow each cell to grow into a clone of the cell).
- Screen supernatant of each clone for the presence of the desired antibody.
- Grow chosen clone of cells in tissue culture indefinitely.
- Harvest antibody from the culture.
Applications of Monoclonal Antibodies
- Diagnostic Applications: (a) Biochemical analysis (b) Diagnostic imaging
- Therapeutic Applications: (a) Direct use of mAB’s as therapeutic agents (b) mAB’s as targeting agents
- Protein Purification
1. Diagnostic Applications:
(a) Biochemical Analysis:
- It is used in the Radioimmuno assays (RIA) and Enzyme-linked Immunosorbent assays (ELISA) in the Laboratory.
- These assays measure the circulating concentration of Hormones (Insulin, HcGHuman Chorionic Gonadotropin, Growth Hormone, Progesterone, Thyroxine, Triiodothyronine, Thyroid Stimulating Hormone) several other tissue and cell products (Blood Group antigen, Blood clotting factors, interferon’s, interleukins, tumor markers).
- Pregnancy by detecting the urinary levels of HcG.
- Hormonal disorders analysis of thyroxine, triiodothyronine.
- Cancer estimation of plasma carcinoembryonic antigen in colorectal cancers and prostate-specific antigen for prostate cancer.
(b) Diagnostic Imaging:
- Radiolabelled in imaging of diseases and this technique is referred to as Immunoscintigraphy. Radioisotopes commonly used for labeling mAB are Iodine- 131 and technetium-99. The mAB tagged with a radioisotope is injected intravenously into the patients.
- These mAB’s localize at specific sites (say a tumor) which can be detected by imaging the Radioactivity.
- Myocardial Infarction, DVT, Atherosclerosis, etc.
2. Direct Use of mAB’s as Therapeutic Agents:
- In destroying disease-causing organisms – mAB’s promote efficient opsonization of Pathogenic organisms (by coating with antibodies) and enhance Phagocytosis.
- In Immunosuppression of Organ Transplantation: In normal medical practice, immunosuppressive drugs such as cyclosporine and prednisolone are administered to overcome the rejection of organ transplantation. Nowadays mAB’s specific to T-Lymphocyte surface antigen is being used for this purpose.
3. Protein Purification:
- mAB’s can be produced for any protein so the produced mAB’s purification is required against which it is raised.
- mAB’s columns can be prepared by coupling them to cyanogen bromide activated sepharose (chromatographic matrix). The immobilized mAB’s in this manner is very useful for the purification of Proteins by the immunoaffinity method.
- There are certain advantages of using mAB’s for protein purification. These include the specificity of the mAB to bid to the desired protein, very efficient elution from the chromatographic column and a high degree of purification.
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