Monoclonal Antibody Production by In-Vivo Methods (Ascites Production)
Our services includes cell line preparation (when provided with Mycoplasma free live cell line) for immunization into 5 Balb/c Mice, all necessary procedures and husbandry for a typical production run which is based on removing ascites fluid a maximum of 3 times per mouse followed by euthanasia and disposal. 3 to 10 ml per mouse can be anticipated.
Below are some considerations for using the ascites method for producing monoclonal antibodies.
- Some hybridoma cell lines do not adapt well to in-vitro conditions; the yields are negative or too low.
- Monoclonal antibodies from mouse ascites fluids are essential for experiments when they are injected into mice to determine which components of the immune system are responding to the antigens, e.g., for studies on allograft rejections, autoimmune diseases, histocompatibility complexes, and monocytic antigens.
- Hybridoma cells can be propagated in culture but do not always replicate the appropriate antibody activity; e.g., the monoclonal antibodies do not have high affinity for or binding capacity for the epitopes of some protein antigens.
- The elution of some monoclonal antibodies from purification columns is especially sensitive to denaturation or proteolysis, resulting in the loss of or significant decreases in antigen-binding activity.
- This loss of native conformation can also elicit unwanted immune responses when the monoclonal antibodies are administered therapeutically to humans in-vivo.
- Monoclonal antibodies specific to both viral proteins and for cells of the immune system are critical to understanding the ways the body fights retroviral infections. Moreover, such studies require extremely high concentrations of monoclonal antibodies that cannot be subjected to treatments that may alter their native structure. Purification by salt precipitation or affinity chromatography is often not suitable at high densities since such conditions can be harsh and result in denaturation of a fraction of the antibodies, loss of reactivity with the antigen, and enhanced immunogenicity of the antibodies leading inevitably to a shortening of antibody retention time after in-vivo administration.
- Serum-free, cell culture methods as yet can not produce sufficient amounts of monoclonal antibodies anti-human isotypes crucial for the evaluation of new vaccines against serious bacterial infections, e.g., H. influenza, type b, causing meningitis in infants and children.
- Several different Monoclonal Antibodies (IgM) used to remove tumor cells from autologous bone marrow are needed in the treatment of neuroblastoma in children. The hybridoma lines cannot be adapted to growth in serum-free medium; therefore, the monoclonal antibodies must be harvested from mouse ascitic fluids in order to assess new approaches to myeloablative therapy of the tumors.
- Culture methods sometimes yield populations of monoclonal antibodies that are glycosolated at positions different from those harvested from mouse ascitic fluids, thereby influencing the antigen binding capacity and the outcome of certain biologically important processes, e.g., phagocytosis (Such differences in reactivity are important to investigators who intend to use the monoclonal antibodies in-vivo in human or animal experiments.).
- When myeloma cells producing monoclonal antibodies are contaminated, they often must be passaged through the mouse.
- The hybridoma cell line will not adapt well to in vitro conditions.
- In applications where several different mouse MAb at high concentrations are required for injection into mice, the in vitro method can be inefficient
- MAb from mouse ascitic fluids might be essential for experiments in which MAb are used in vivo in mice
- Rat hybridoma cell lines do not generate ascites efficiently in rats, and usually adapt poorly to in vitro conditions, but usually generate ascites in immunocompromised mice.
- Downstream purification can lead to protein denaturation and decreased antibody activity.
- Serum free or low serum conditions cannot provide sufficient amounts of MAb for some purposes, such as the evaluation of new vaccines against infectious organisms.
- Culture methods sometimes yield populations of IgG MAb that are glycosylated at positions different from those harvested from mouse ascites fluid, thereby influencing antigen
- Binding capacity and important biologic functions.
- When hybridoma cells producing MAb are contaminated with infectious agents, such as yeasts or fungi, the cells often must be passed through mice.
- Some cell lines that do adapt to tissue culture conditions become unable to maintain adequate production of MAb