A) How would you predict and find out if a molecule is immunogenic? B) Antibody-
ID: 57427 • Letter: A
Question
A) How would you predict and find out if a molecule is immunogenic?
B) Antibody-based immunoassays are generally useful for detection of pesticide contamination of food products and ground water. A biotech company is interested in developing an immunoassay test kit for a herbicide WONDER G, and you have been asked to help develop an antibody to WONDER G. Outline the strategy and protocol you would follow to prepare a monoclonal antibody specific for WONDER G, a molecule with a molecular weight of 2500 (Dalton). How could you be sure your antibody is specific for WONDER G?
C) What are some advantages of this monoclonal antibody (produced by a hybridoma) over a polyclonal antibody preparation also reactive with WONDER G?
Explanation / Answer
A. A immunogen is a molecule which is capable of inducing an immune response. There are certian requirements for a molecule to be immunogen like high molecular weight, foreignness, chemical complexicity and degradability.
To identify the molecule as immunogen, we need to inject the molecule into a mice or rabbit and have to check its immune response by antibody titers in the sera. A good immunogen will elicit a strong antibody response at very low dilution of sera. This can be done using Enzyme Linked Immuno Sorbent Assay (ELISA).
B. These antibodies are produced by cell lines or clones obtained from animals that have been immunized with the substance that is the subject of study. To produce the desired mAb, the cells must be grown in either of two ways: by injection into the abdominal cavity of a suitably prepared mouse or by tissue culturing cells in plastic flasks. Further processing of the mouse ascitic fluid and of the tissue culture supernatant might be required to obtain mAb with the required purity and concentration.
Step 1: Immunization of Mice and Selection of Mouse Donors for Generation of Hybridoma Cells
Mice are immunized with an WONDER G that is prepared for injection either by emulsifying with Freund's adjuvant or other adjuvants or by homogenizing a gel slice that contains the antigen. In almost all laboratories, mice are used to produce the desired antibodies. In general, mice are immunized every 2-3 weeks but the immunization protocols vary among investigators. When a sufficient antibody titer is reached in serum, immunized mice are euthanized and the spleen removed to use as a source of cells for fusion with myeloma cells.
Step 2: Screening of Mice for Antibody Production
After several weeks of immunization, blood samples are obtained from mice for measurement of serum antibodies. Several humane techniques have been developed for collection of small volumes of blood from mice. Serum antibody titer is determined with various techniques, such as enzyme-linked immunosorbent assay (ELISA) and flow cytometry. If the antibody titer is high, cell fusion can be performed. If the titer is too low, mice can be boosted until an adequate response is achieved, as determined by repeated blood sampling. When the antibody titer is high enough, mice are commonly boosted by injecting antigen without adjuvant intraperitoneally or intravenously (via the tail veins) 3 days before fusion but 2 weeks after the previous immunization. Then the mice are euthanized and their spleens removed for in vitro hybridoma cell production.
Step 3: Preparation of Myeloma Cells
Fusing antibody-producing spleen cells, which have a limited life span, with cells derived from an immortal tumor of lymphocytes (myeloma) results in a hybridoma that is capable of unlimited growth. Myeloma cells are immortalized cells that are cultured with 8-azaguanine to ensure their sensitivity to the hypoxanthine-aminopterin-thymidine (HAT) selection medium used after cell fusion.1 A week before cell fusion, myeloma cells are grown in 8-azaguanine. Cells must have high viability and rapid growth. The HAT medium allows only the fused cells to survive in culture.
Step 4: Fusion of Myeloma Cells with Immune Spleen Cells
Single spleen cells from the immunized mouse are fused with the previously prepared myeloma cells. Fusion is accomplished by co-centrifuging freshly harvested spleen cells and myeloma cells in polyethylene glycol, a substance that causes cell membranes to fuse. As noted in step 3, only fused cells will grow in the special selection medium. The cells are then distributed to 96 well plates containing feeder cells derived from saline peritoneal washes of mice. Feeder cells are believed to supply growth factors that promote growth of the hybridoma cells. Commercial preparations that result from the collection of media supporting the growth of cultured cells and contain growth factors are available that can be used in lieu of mouse-derived feeder cells. It is also possible to use murine bone marrow-derived macrophages as feeder cells.
Step 5: Cloning of Hybridoma Cell Lines by “Limiting Dilution” or Expansion and Stabilization of Clones by Ascites Production
At this step new, small clusters of hybridoma cells from the 96 well plates can be grown in tissue culture followed by selection for antigen binding or grown by the mouse ascites method with cloning at a later time. Cloning by “limiting dilution” at this time ensures that a majority of wells each contain at most a single clone. Considerable judgment is necessary at this stage to select hybridomas capable of expansion versus total loss of the cell fusion product due to underpopulation or inadequate in vitro growth at high dilution. In some instances, the secreted antibodies are toxic to fragile cells maintained in vitro. Optimizing the mouse ascites expansion method at this stage can save the cells. Also, it is the experience of many that a brief period of growth by the mouse ascites method produces cell lines that at later in vitro and in vivo stages show enhanced hardiness and optimal antibody production.
C. The advantages of monoclonal antibodies over polyclonal