Rabbit hybridoma
Encyclopedia
A rabbit hybridoma is a hybrid cell line formed by the fusion of an antibody producing rabbit B cell
with a cancerous B-cell (myeloma).
and more diverse recognition of many molecules including phospho-peptides, carbohydrates and immunogens that are not otherwise immunogenic
in mouse. However, until recently, the type of antibodies available from rabbit had been limited in scope to polyclonal antibodies. Several efforts were made to generate rabbit monoclonal antibodies
after the development of mouse hybridoma technology in the 1970s. Research was conducted into mouse-rabbit hetero-hybridomas to make rabbit monoclonal antibodies. However, these hetero-hybridomas were ultimately difficult to clone, and the clones, generally unstable, and did not secrete antibody over a prolonged period of time.
from a rabbit that has been immunized. There are numerous immunization protocols for rabbit, notably for the generation of polyclonal antibodies. After immunization, B-cells are fused with a candidate rabbit fusion partner cell line to form hybridomas. Resulting antibodies from hybridomas are screened for an antigen which meets criteria of interest by diagnostic tests such as ELISA
, Western blot
, Immunohistochemistry
and FACS. The resulting hybrdomas may be subcloned to ensure monoclonal characteristics.
B cell
B cells are lymphocytes that play a large role in the humoral immune response . The principal functions of B cells are to make antibodies against antigens, perform the role of antigen-presenting cells and eventually develop into memory B cells after activation by antigen interaction...
with a cancerous B-cell (myeloma).
History
The rabbit immune system has been documented as a vehicle for developing antibodies with higher affinityAffinity
Affinity is a word used in a variety of fields, usually to indicate some kind of preference, relationship, or a potential or actual closeness between two entities.Articles dealing with various usages of the word: affinity include:-Commerce and law:...
and more diverse recognition of many molecules including phospho-peptides, carbohydrates and immunogens that are not otherwise immunogenic
Immunogenicity
Immunogenicity is the ability of a particular substance, such as an antigen or epitope, to provoke an immune response in the body of a human or animal.- Immunogenicity :The ability to induce humoral and/or cell-mediated immune responses....
in mouse. However, until recently, the type of antibodies available from rabbit had been limited in scope to polyclonal antibodies. Several efforts were made to generate rabbit monoclonal antibodies
Monoclonal antibodies
Monoclonal antibodies are monospecific antibodies that are the same because they are made by identical immune cells that are all clones of a unique parent cell....
after the development of mouse hybridoma technology in the 1970s. Research was conducted into mouse-rabbit hetero-hybridomas to make rabbit monoclonal antibodies. However, these hetero-hybridomas were ultimately difficult to clone, and the clones, generally unstable, and did not secrete antibody over a prolonged period of time.
Initial fusion partner
In 1995, Katherine Knight and her colleagues, at Loyola University of Chicago, succeeded in developing a double transgenic rabbit over-expressing the oncogenes v-abl and c-myc under the control of the immunoglobulin heavy and light chain enhancers. The rabbit formed a myeloma-like tumor, allowing the isolation of a plasmacytoma cell line, named 240E-1. Fusion of 240E-1 cells with rabbit lymphocytes produced hybridomas that secreted rabbit monoclonal antibodies in a consistent manner. However, like the early mouse myeloma lines developed in 1970s, stability was a concern. A number of laboratories which had received the 240E-1 cell line from Dr. Knight’s laboratory reported stability problems with the fusion cell line 240E-1.Improved fusion partner
In 1996, Weimin Zhu and Robert Pytela, at the University of California San Francisco (UCSF), obtained 240E-1 from Dr. Knight’s laboratory and attempted to develop an improved rabbit hybridoma. Improvements in the characteristics of 240E-1 were accomplished by repeated subcloning, selection for high fusion efficiency, robust growth, and morphological characteristics such as a bright appearance under a phase contrast microscope. Selected subclones were further tested for their ability to produce a stable hybridoma and monoclonal antibody secretion. After multiple rounds of subcloning and selection processes, a new cell line named 240E-W, was identified and which expressed better fusion efficiency and stability. Cell line 240E-W has since been further developed and optimized for production of rabbit monoclonal antibodies for research and commercial applications.Process
The process of hybridoma formation in a rabbit first entails obtaining B-cellsB cell
B cells are lymphocytes that play a large role in the humoral immune response . The principal functions of B cells are to make antibodies against antigens, perform the role of antigen-presenting cells and eventually develop into memory B cells after activation by antigen interaction...
from a rabbit that has been immunized. There are numerous immunization protocols for rabbit, notably for the generation of polyclonal antibodies. After immunization, B-cells are fused with a candidate rabbit fusion partner cell line to form hybridomas. Resulting antibodies from hybridomas are screened for an antigen which meets criteria of interest by diagnostic tests such as ELISA
ELISA
Enzyme-linked immunosorbent assay , is a popular format of a "wet-lab" type analytic biochemistry assay that uses one sub-type of heterogeneous, solid-phase enzyme immunoassay to detect the presence of a substance in a liquid sample."Wet lab" analytic biochemistry assays involves detection of an...
, Western blot
Western blot
The western blot is a widely used analytical technique used to detect specific proteins in the given sample of tissue homogenate or extract. It uses gel electrophoresis to separate native proteins by 3-D structure or denatured proteins by the length of the polypeptide...
, Immunohistochemistry
Immunohistochemistry
Immunohistochemistry or IHC refers to the process of detecting antigens in cells of a tissue section by exploiting the principle of antibodies binding specifically to antigens in biological tissues. IHC takes its name from the roots "immuno," in reference to antibodies used in the procedure, and...
and FACS. The resulting hybrdomas may be subcloned to ensure monoclonal characteristics.