Transcript elisa
Monoclonal Antibody Prepared by: AMER SAAD AL-ALI ABDUALLAH SAUD AL-SHETELY TAREQ NAFEA AL-HARBY Discovery In the 1970s, the B-cell cancer myeloma was known, and it was understood that these cancerous B-cells all produce a single type of antibody. This was used to study the structure of antibodies, but it was not possible to produce identical antibodies specific to a given antigen. The process of producing monoclonal antibodies invented by Georges Köhler and César Milstein in 1975; they shared the Nobel Prize in Physiology or Medicine in 1984 for the discovery. The key idea was to use a line of myeloma cells that had lost their ability to secrete antibodies, and come up with a technique to fuse these cells with healthy antibody producing B-cells. Definition MCA are antibodies that are identical because they were produced by one type of immune cell (B cell), all clones of a single parent cell. Given any substance, it is possible to create monoclonal antibodies that specifically bind to that substance; they can then serve to detect or purify that substance. This has become an important tool in biochemistry, molecular biology and medicine. PRODUCTION OF MONOCLONAL ANTIBODY HYBRIDOMA TECHNOLOGY PRODUCTION OF MONOCLONAL ANTIBODY HYBRIDOMA TECHNOLOGY Step 1: - Immunization Of Mice & Selection Of Mouse Donor For Generation Of Hybridoma cells ANTIGEN ( Intact cell/ Whole cell membrane/ micro-organisms ) + ADJUVANT (emulsification) Ab titre reached in Serum Spleen removed (source of cells) PRODUCTION OF MONOCLONAL ANTIBODY HYBRIDOMA TECHNOLOGY Step 2: - Screening Of Mice For Antibody Production After several weeks of immunization Serum Antibody Titre Determined (Technique: - ELISA / Flow cytometery) Titre too low BOOST (Pure antigen) Titre High BOOST 2 weeks (Pure antigen) PRODUCTION OF MONOCLONAL ANTIBODY HYBRIDOMA TECHNOLOGY Step 3: - Preparation of Myeloma Cells + 8 - Azaguanine Myeloma Cells Immortal Tumor Of Lymphocytes Myeloma Cells HGPRTHigh Viability & Rapid Growth PRODUCTION OF MONOCLONAL ANTIBODY HYBRIDOMA TECHNOLOGY Step 4: - Fusion of Myeloma Cells with Immune Spleen Cells & Selection of Hybridoma Cells PEG FUSION SPLEEN CELLS MYELOMA CELLS Feeder Cells Growth Medium 1. HYBRIDOMA CELLS ELISA PLATE 2. HAT Medium Plating of Cells in HAT selective Medium Scanning of Viable Hybridomas PRODUCTION OF MONOCLONAL ANTIBODY HYBRIDOMA TECHNOLOGY Step 5: - Cloning of Hybridoma Cell Lines by “ Limiting Dilution” or Expansion A. Clone Each +ve Culture B. Test Each Supernatant for Antibodies C. Expand +ve Clones Tissue Culture Method Mouse Ascites Method PRODUCTION OF MONOCLONAL ANTIBODY HYBRIDOMA TECHNOLOGY Mca.swf Applications of Monoclonal Antibodies Diagnostic Applications Biosensors & Microarrays Therapeutic Applications Transplant rejection Cardiovascular disease Cancer Infectious Diseases Inflammatory disease Clinical Applications Purification of drugs, Imaging the target Future Applications Fight against Bioterrorism Monoclonal antibodies for cancer treatment Three mechanisms that could be responsible for the cancer treatment. A. mAbs act directly when binding to a cancer specific antigens and induce immunological response to cancer cells. Such as inducing cancer cell apoptosis, inhibiting growth, or interfering with a key function. B. mAbs was modified for delivery of a toxin, radioisotope, cytokine or other active conjugates. C. it is also possible to design bispecific antibodies that can bind with their Fab regions both to target antigen and to a conjugate or effector cell mAbs treatment for cancer cells ELISA Enzyme Linked Immunosorbent Assay What is ELISA? ELISA is a biochemical technique used mainly in immunology to detect the presence of an antibody or an antigen in a sample. The ELISA has been used as a diagnostic tool in medicine and plant pathology. The principle of (ELISA) It is a solid phase assay that requires the separation of reagents. The principle depend on the type of ELISA(direct or indirect) Applications Because the ELISA can be performed to evaluate either the presence of antigen or the presence of antibody in a sample, it is a useful tool both for determining serum antibody concentrations (such as with the HIV test) and also for detecting the presence of antigen. It has also found applications in the food industry in detecting potential food allergens such as milk, peanuts and eggs. The ELISA test, or the enzyme immunoassay (EIA), was the first screening test commonly employed for HIV. It has a high sensitivity. In an ELISA test, a person's serum is applied to a plate to which HIV antigens have been attached. The plate is then washed to remove other components of the serum. Then an antibody applied to the plate, followed by another wash. This antibody is chemically linked in advance to an enzyme. A substrate for the enzyme is applied, and catalysis by the enzyme leads to a change in color or fluorescence. ELISA results are reported as a number either positive or negative result. Positive Negative Control Control 1.689 0.153 Patient A Patient B Patient C O.055 0.412 1.999 Assay Control 0.123 Above is ELISA data from three patients. Numbers are expressed as optical density at 450 nm: Positive result ≥0.500. 0.300 to 0.499 are indeterminate and need to be retested. Negative ≤0.300. Anim0100.swf Reference MedLinePlus. "HIV ELISA/western blot." U.S. National Library of Medicine. Last accessed April 16, 2007 . http://www.nlm.nih.gov/medlineplus/ency/article/003538.htm Lequin R (2005). "Enzyme immunoassay (EIA)/enzyme-linked immunosorbent assay (ELISA).". Clin. Chem. 51 (12): 2415-8. PMID 16179424. Köhler, G., and Milstein, C. Continuous cultures of fused cells Secreting antibodies of predefined specificity. Nature, 256: 495, (1975). Koprowski, H., Steplewski, Z., Herlyn, D., and Herlyn, M. Production of monoclonal antibody against human melanoma by somatic cell hybrids. Proc. Nat. Acad. Sci. USA. 75: 3405, (1978).