Definition: Mono: One Clone: A strain of cells descended form a single cell Antibody: A molecule of animal origin that has immunolgical activity only against the antigen to which it was made
What’s an ANTIGEN? Antigens are soluble substances They may be toxins or foreign proteins They may be whole cells OR just parts of a cell, like a component of the cell wall Proteins usually make the best antigens Very small proteins (peptides) can be coupled (attached) to carriers so they make better antigens
How to make a monoclonal antibody? Step 1: Immunize mice Step 2: Test the serum Step 3: Perform a fusion Step 4: Screen the fusion for the right cells Step 5: Grow the hybridomas Step 6: Harvest the antibody Step 7: Concentrate and purify the product
Step 1: Immunize the mice - Housing Can uses Balb C mice to make monoclonals. The mice are housed in a laminar flow hood to minimize their exposure to other antigens. Each box contains mice for only one project.
Step 1: Immunize the mice - Identification Each mouse has a microchip implanted to identify it. Each time the mouse is inoculated or bled, the chip is checked.
Step 1: Immunize the mice - Inoculation The mice are aseptically inoculated with the antigen combined with an adjuvant. Inoculations are done either sub-cutaneously or intra- peritoneally. Normal dose per mouse is between 50 and 100 micrograms of protein. Inoculations are performed every 21 to 28 days.
Step 2: Test the serum - Bleeding the mice Blood is drawn into the tube. A capillary tube is applied to nicked vein.
Step 2: Test the serum - In the Lab Whole blood from the immunized mice is transported to the lab. The whole blood is spun down in a centrifuge to separate the serum. The serum is diluted in a series from 1:300 to 1:300,000.
Step 2: Test the serum - ELISA E L IS A Diluted serum is tested by an ELISA (Enzyme Linked ImmunoSorbent Assay) Color change in the ELISA substrate indicates the amount of antibody present in the diluted serum.
Step 2: Test the serum - Decision time When the serum titer of the mice has reached a plateau, an additional ELISA test is performed to determine the predominant isotype present. The two isotypes that are most common in mouse serum are IgG and IgM. Sometimes additional testing is done (Western blots, immunoflurorescence) to determine whether the serum response is specific for the selected antigen. The mouse with the strongest, most specific response is chosen for the fusion.
Step 3: Perform a fusion - Feeder Plates 1) Euthanize an ICR mouse. 2) Aseptically open the peritoneal cavity of the mouse. 3)Use sterile media and a pipet to flush macrophage cells from the peritoneal cavity. 4) Dilute the macrophage cells in media. 5) Distribute the diluted macrophage cells into 96-well plates. 6) Allow the feeder cells to grow for 24 hours.
Step 3: Perform a fusion - Prepare spleen cells 1) Anesthetize the best responder among the immunized mice. 2) Perform a cardiac puncture and withdraw whole blood from the mouse. 3) Euthanize the mouse. 4) Aseptically open the mouse’s abdomen. 5) Dissect away the peritoneal membrane to expose the spleen. 6) Carefully remove the spleen to a sterile petri dish. 7) Make a single cell suspension of the spleen.
Step 3: Perform a fusion - Hybridomas 1) Dilute the spleen cells. 2) A special line of tumor cells (SP 2/0) has already been grown in a flask. 3) Mix the spleen cells with the tumor cells. 4) Add a chemical agent to the mixture to “soften” the cell membranes. 5) Place the now-fused spleen/tumor cells, called hybridomas, on top of the feeder cell layer in the 96-well plate.
Step 3: Perform a fusion - Growing the cells (hybridomas) Fusions are performed in a lab. Cells are grown in 96-well plates.
Step 3: Perform a fusion - Growing the cells (hybridomas) Cells are grown in a 37 o C incubator. Cells are kept in an atmosphere of about 6% CO 2. The cells are fed after 7 days of incubation. The cells are checked for growth after 10 days of incubation.
Step 4: Screen the fusion for the right cells 1) When growth is detected in one of the wells of the 96-well plate, cell supernatant from that well is tested by ELISA. 2) If the well tests positive, cells from it are removed and diluted. The diluted cells are plated into new 96-well plates at a dilution that will contain only one cell per well. 3) The wells are tested again and positive wells are re- diluted. This process is called cloning. 4) The object of cloning is to obtain a population of identical cells all producing the same antibody - monoclonal cells.
Step 5: Grow the hybridomas - 24-well plates Once a monoclonal population of cells is obtained, they are expanded to produce the desired amount of antibody. The antibody is produced by the cells and released into the cell supernatant. First, the monoclonal cells are expanded into 24-well plates.
Step 5: Grow the hybridomas - Flasks Then, when the cells in the 24-well plates are growing well and look healthy, they are transferred into flasks. Cells in the flasks are expanded, as needed. Some cells are preserved for future use by freezing.
Step 6: Harvest the antibody 1) The antibody is produced by the cells and released into the cell supernatant. 2) Media is added to the flasks until the desired volume of antibody- containing supernatant is obtained. 3) When the desired volume is reached, the supernatant is refrigerated and tested by ELISA. 4) If the testing is successful, the supernatant can be used by the investigator, or concentrated and purified.
Step 7: Concentrate and Purify the Product The antibody can be concentrated by precipitation with a solution of saturated ammonium sulfate. Concentrated antibody can then be purified by passing it through a column.
So, why make monoclonal antibodies? Monoclonal antibodies can be used in three general ways: to purify reagents for tests and research as labeling agents for detection assays for experimental therapy.
More specifically, monoclonal antibodies can: Be attached to a color agent of fluorescent chemical for immunological staining. Be attached to a medium in a column to purify other substances that will bind to them (affinity purification). Coat plates for ELISA testing, as for a diagnostic test. Be used as therapy by attaching to a particular target cell and marking it for lysis by natural killer cells or medicines Be radiolabeled (attached to an isotope) for diagnostic imaging such as PET (Posirton Emission Technology)