61 terms

Hybridoma technology

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What is the major obstacles hampering progress in antibody research and use of anti-serum?
Antibody production in sheep and horses are polyclonal so concentrations change over time
What are monoclonal antibodies?
Antibodies with identical isotypes, affinity and avidity that are produced in the laboratory by cells derived from a single B cell or plasma cell
How are monoclonal antibodies created?
1. a mouse is injected with a specific antigen to make it produce antibodies
2. the spleen is removed from the mouse in order to obtain B cells producing specific antibody
3. a hybrid cell is cultured that is made form myeloma cells and B cells
What are the problems with polyethylene glycol fusion?
There are multiple possible outcomes besides just the Spleen-SP2/0 fusion hybrid cell
What are the possible outcomes of polyethylene glycol?
Spleen cells
Sp2/0 myeloma cells
Spleen cell-spleen cell fusion
Sp2/0-SP2/0 fusion
Spleen-SP2/0 fusion
How are spleen-SP2/0 fusions selected?
HAT selection
What three components make up HAT
Hypoxanthanine
Aminopterin
Thymidine
What are the two different nucleic acid pathways?
De novo synthesis
Salvage pathway
What does the De novo synthesis pathway require?
Viable folic acid pathway
What does the Salvage pathway require?
Thymidine and Hypoxanthine
What compound blocks the De Novo pathway?
Aminopterin
What component is necessary for the De Novo pathway?
Tetrahydrofolic pathway
Phosphoribosyl pyrophosphate and Uridylate are used in what pathway?
De Novo Pathway
Thymidine (TK+) and HGPRT+ are used in what pathway?
Salvage Pathway
What pathway(s) do you use to get only SP2/0-SP2/0 fusions?
De novo synthesis only
What pathway(s) do you use to get SP2/0-SP2/0 fusion cells and Spleen-SP2/0 fusion cells?
De novo synthesis pathway and the Salvage pathay
How do you tell which fusion products are producing antibody?
Place cells in a medium that only allows the hybrid cells to proliferate
What is a Hybridoma?
Hybrid cell line obtained by fusing a lymphoid tumor cell with a lymphocyte which then has both the immortality of the tumor cell and the effector function of the lymphocyte
What are the advantages of hybridomas producing monoclonals?
1. Infinite source of monoclonal antibodies
2. Identical affinity and avidity?
3. Identical affinity constant?
4. Single isotype
Human
u
Mouse
o
Rat
a
Humanized
zu
Hamster
e
Primate
l
Chimera
xi
Viral
vir
Bacterial
bac
Immune
lim
Tumors
tum
Monoclonal antibody
mab
What is the major problem with murine monoclonals as a therapeutic agent?
They reduce monoclonal antibodies that are antigenic
How do you circumvent the foreign protein issue?
Chimeric molecules
Grafted antibody combining sites
Transgenic animals producing human antibodies
What is XenoMouse?
a mouse strain engineered to produce human antibodies in the place of mouse antibodies by introduction of magabase-sized fragments of the human heavy and kappa light chain loci into the germ line of mice deficient in mouse antibody production
Hybridomas were shown to be stable and to produce significant levels of human monoclonal antibodies
What are Naked antibodies directed towards?
CD20 and CD52
What is Rituximab (Rituxan) used for?
Used to treat B-cell non-Hodgkin lymphoma and some other diseases. It is a monoclonal antibody against the CD20 antigen, found on B cells
What is Alemtuzumab (Campath) used for?
is an antibody against the CD52 antigen, which is present on both B cells and T cells, It is used to treat some patients with B-cell chronic lymphocytic leukemia
What is MAb effect on tumor cells?
specifically targets tumor antigen cells. target tumor
What are Avidin systems?
Structures that bind to biotin and then binds to an antibody before entering the cell
What are antibody drug conjugates?
When a drug is attached to a monoclonal antibody that is directed toward a tumor cell
What is a tri specific antibody?
An antibody with three receptor regions
What are the two radiolabled antibodies that have been approved to treat cancer?
Ibritumomab tiuxetan (Zevalin)
Tositumomab (Bexxar)
How does Zevalin work and what is it used for?
delivers radioactivity directly to cancerous B lymphocytes. It is used to treat B-cell non-Hodgkin lymphoma that has not responded to standard treatment
What is Bexxar used to treat?
used to treat certain types of non-Hodgkin lymphoma that no longer responds to Rituxan or chemotherapy
How does Muromonab-CD3 (OKT3) immunosuppress?
With two humanized anti-CD3 monoclonals it binds to CD3 molecules on the surface of T cells. Used to prevent acute rejection of organ transplant
How does Inflixmab (Remicade) immunosuppress?
Binds to tumor necrosis factor alpha. shows promise in the treatment of rheumatoid arthritis
How does Omalizumab (Xolair) immunosuppress?
Binds to IgE preventing IgE from binding to mast cells. shows promise against allergic asthma
How does Daclizumab (Zenapax) immunosuppress?
Binds to part of the IL-s receptor produced at the surface of activated T cells. Used to prevent acute rejection of transplanted kidneys
How does Herceptin (trastuzumab) stop cancer cells?
Binds HER2, a receptor for epidermal growth factor that is found on some tumor cells. Only monoclonal that is effective against solid tumors
How does Erbitux (cetuximab) stop cancer cells?
Blocks HER1, another EGF receptor
Drugs used for immunosuppression
Muromona-CD3
Inflixmab
Omalisumab
Daclizumab
Drugs used for Oncogene inactivation
Herceptin
Erbitux
Drugs used for monoclonals and malignannt cells
Rituximab
Zevalin
Bexxar
How does Rituximab treat monoclonal and malignant cells?
Binds to the CD20 molecule found on most B cells and is used to treat B cell lymphomas
How does Zevalin treat monoclonal and malignant cells?
This is a monoclonal antibody against the CD20 molecule on B cells conjugated to either the radioactive isotope indiumm-112 or the radioactive isoptope yttrium-90
How does Bexxar treat monoclonal and malignant cells?
A conjugate of a monoclonal antibody against CD20
Drugs that are used for angiogenesis inhibitors
Vitaxin
Becacizumab
How is Vitaxin a angiogenesis inhibitor?
Binds to a vascular intergrin found on the blood vessels of tumors but not on the blood vessels supplying normal tissues
How is a Becacizumab angiogenesis inhibitor?
Binds to VEGF preventing it from binding to its receptor
Identification of lymphocyte subsets
Flow cytomerty
think pregnancy test
Zmapp
Mouse human chimeric antibodies
directed toward Ebola surface gylcoprotein
produced in tobacco plants
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